Cliffjumper's death in the first episode. Especially the Autobot's reactions (special mention to Arcee) and Optimus' speech in his memory. It's very clear that he's trying to keep Arcee from running off and doing something stupid, and every iota of sadness and pain is clearly distinguishable in his voice.
Optimus: We must not allow our anger over the loss of Cliffjumper to impair our judgement. As of today, only we five Autobots remain on this earth. We owe it to ourselves... to the memory of Cybertron... to any Autobots in any galaxy seeking safe harbor... to humankind... and we owe it to the memory of our fallen comrade... to survive.
Skyquake's death in "Masters and Students." We see a shot of one of his eyes closing for the last time before a visibly saddened Optimus really drives it home.
Optimus: Had Skyquake chosen to stray from his master's path... we might not be burying him today.
In "Scrapheap", Arcee and Optimus are stuck dying of robo-hypothermia in the Arctic. As they begin to succumb to the cold, Arcee reaches up and holds Optimus's hand.
Arcee's terrifying flashback in "Predatory" of Tailgate's murder at the hands of Airachnid, especially the part when she helplessly cries his name after he dies.
Miko and Bulkhead trapped underground in "Rock Bottom", with Miko having a major breakdown.
In particular, seeing Miko rendered speechless out of worry, fear and sheer exhaustion is particularly touching, even for some viewers who don't like her.
Arcee talking to her dead partner Cliffjumper at the start of "Partners".
Arcee discovering that Starscream was the one who murdered Cliffjumper.
From the same episode, Bumblebee's look of sheer disappointment upon seeing Arcee about to kill Starscream.
Episode 21: Miko, first when she realizes that Bulkhead's losing his memory of her, and later when it looks like he's been left brain dead.
Episode 22- Ratchet confessing his sins while almost bleeding to death. Much crying was had by all.
Ratchet: Optimus... I've been a fool...
Raf being held on life support after being shot by Megatron. He was teetering on the edge of life.
Bumblebee's face when he saw him was heartbreaking. Not to mention it's one of the few times in which he almost rushes out to try and kill Megatron for doing it.
Add to that how Ratchet berates himself for being too prideful to bother learning about human science and medicine as he cannot find a way to treat Raf, and then pretty much confessing how attached he's gotten to Raf, which could also be seen in the previous episode, when Ratchet invited Raf to watch as he tested synthetic energon.
Bumblebee and Raf looking sadly at one another as Raf is driven away in "One Shall Rise, Part 1" is quite heart-wrenching. Thankfully, it's not too long before they're reunited.
The end of "One Shall Rise, Part 3". For using the Matrix to take Unicron down, Optimus is completely unable to remember his life, save for his time as Orion Pax. Megatron tells him the Autobots are his mortal enemies and then summons a GroundBridge to the Nemesis. Before Optimus goes through, he sees the faces of the other Autobots, and is completely unable to recognise them. Bumblebee's face is once again the saddest part.
Especially the way Bumblebee hopelessly beeps "Optimus!"
Seeing the Band of Brothersbroken into an arguing mess after losing Optimus is a tear jerker in a "man, they really did need him" sort of way.
Arcee's reaction to being back on her dead homeworld of Cybertron and seeing the ruins of its pre-war civilization was extremely powerful.
Arcee: This isn't how I wanted you to see my home.
The way Orion Pax is treated throughout "Orion Pax, Part 3", culminating in him being beaten up by two Vehicons who talk about how good it feels, while he's preaching to their better nature. This leads to a Moment of Awesome (and a possible Moment Of Funny) when Orion realizes that he has guns and kills the Vehicons.
To elaborate on his treatment a little further: he's eventually forced to work at gunpoint, tries to reason with the Vehicons for the sake of others' safety, gets beaten up for his efforts and doesn't fight back (believing himself to be unarmed), then briefly pleads with them to stop before he finds his guns. Simply seeing the normally strong, in-control and completely badass leader of the Autobots reduced to begging fearfully, however briefly, will certainly strike the heart of any Transformers fan.
Ratchet telling Raf about how the field medic who saved Bumblebee's life after Megatron tore out his voicebox and left him for dead wasn't able to fix Bumblebee's voice. Of course, he's speaking about his OWN inability to completely fix Bumblebee, since he was the "field medic" who saved Bee's life.
Ratchet: Yes, well... the medic could have done better.
Hell, Bumblebee's depression was quite sad too.
Followed by Ratchet's heartwrenching plea to donate his T-Cog to Bumblebee, saying he rarely needs it anyways. Both the viewers and Optimus instantly see this as Ratchet's way of trying to atone for not being able to fix Bumblebee before.
Bumblebee's utter despair at his T-cog being destroyed... initially.
A subtle one in "Loose Cannons" when Bulkhead has a Hope Spot that another Wrecker is alive. Then Wheeljack says that Dreadwing killed him.
"Crossfire" gives us Breakdown's death, which manages to be horrific and heartbreaking at the same time. An Insecticon pins his arm with webbing and Airachnid viciously tears him apart offscreen. What makes it so sad is that Breakdown was a relatively decent villain who treated his underlings well and, while a boisterous brute, generally avoided kicking the dog. The worst part of all, though, is that MECH is almost certainly going to use his remains for Project Chimera. Even he deserved better than that.
Optimus being gutted by Silas controlling Nemesis Prime. Thank the Matrix he recovered.
In "Toxicity", Bulkhead slowly dying while trying to destroy the Tox-En throughout the episode can be pretty hard to watch. Also Hardshell's seemingly fatal attack at the end, leaving his fate uncertain.
Bulkhead's flashbacks. Dear lord, the flashbacks. Almost makes you believe that this will be his last episode.