Yusuf's death. It's unexpected, and is made even worse by the fact that Yusuf is a very likeable character, and does a lot to support and help the player.
Clay's death. A double whammy, really. First, you (the player) are led to believe he has betrayed you and started to actively attempt to murder you. Then you discover that this is not the case, and the reason behind all this glitching and Animus Island being deleted with you still on it is actually the Animus itself. And then he sacrifices himself to save you.
Even sadder considering that Sixteen has been a true friend and guide to Desmond throughout the entire series.
The third Alta´r memory with Maria's death and Alta´r's exile.
The final sequence where Ezio discovers Alta´r's final resting place and the final Masyaf key. Manly tears were shed. "Sit a moment and rest" indeed...
In the The Lost Archive DLC, Clay's relationship with his dad. His dad's a total jerk to him, but Clay loves him throughout, even sending him an e-mail from inside the Animus telling him it's not his fault that Clay died.
The Lost Archive as a whole is a very sober story. The entire DLC is a big tearjerker since you're playing through Clay's life, right until his death. Here's a character we hardly knew and could barely trust or understand when we first meet him face to face (or data to data). Then we learn how in youth he wanted to be an astronaut, but in the end became an Assassin, and how he tries hard to care for his father who finds Clay isn't doing good enough, and just shortly before he became Test Subject 16 his mother kills herself by intentionally driving while intoxicated. Doing the whole DLC, we suddenly get why Clay seems so abrasive to Desmond in Revelations. His encounter with Juno was basically "you're not the guy we need, but you're going to help us help him", and his reception of first hearing that was that he felt jaded. Then after Lucy's betrayal Clay is dejected and concedes, muttering a weak "I will" in response to Juno's command. The DLC makes sure you know when what has happened, from the bleeding effect, to when Desmond enters the story, and to when Clay dies; it's a dock with a boat manned by Charon, the ferryman of the dead in Greek mythology. And in the end we're in the clouds under a blue sky and we finally see the exit; it's the Animus loading room beyond that archway. But we know by now that Clay's body is "worm food now", and the Animus detects it. The bridge collapses and he falls back to the beginning.
The E3 trailer (see above), though the extended version of the trailer (acting as the opening cutscene for Ezio's first Sequence) revealed that Ezio was not hanged at Masyaf and managed to escape.
Seeing Ezio as an old man in Assassin's Creed: Embers. He's not old in the grey-bearded, still-as-agile-as-he-was-as-a-teenager way he is in Revelations, but in the longing for his youth, suffering chest pains and getting patted on the head in a slightly patronizing way by his daughter/wife way.
The sneak peek of Assassin's Creed Embers. We see Ezio leading a peaceful life and his strong and loving relationship with his playful daughter. But hearing his constant coughing and short breaths as he walks, let alone his pained breathing as he runs frantically towards his daughter after he sees and mistakes Shao Jun as an enemy, it really hits home just how little time he really does have left. In the end, after a heavy bout of chest pains and coughing, Ezio's last moment is to look at his own smiling wife and daughter before he passes away on a bench in the Piazza della Signoria... where his father and brothers had been executed almost fifty years before.
In an otherwise throwaway line between Sequences, Subject 16 causes a brief Heroic BSOD for Desmond when telling him not to be late for "Lucy's funeral!" Compounded when Shaun mentioned that she had already been quietly buried in a little cemetery outside Rome, and when told by Rebecca or Shaun that she did have feelings for Desmond. After The Reveal that this character is also The Mole and the reason for 16's condition, however, the sadness about this goes away.
Alta´r's memories, where we find out that his late life was less than perfect:
The third one: While Alta´r had ventured east to deal with Genghis Khan, Alta´r's youngest son was wrongfully executed and told that his own father had ordered it. Alta´r came back to Masyaf only to be betrayed by his own brotherhood and lose Maria, when she put herself between him and one of Abbas' loyalists that Alta´r was using the Apple on, only for that loyalist to stab Maria in the back in his death throes. It's even more heartbreaking if you've read the version of this event in The Secret Crusade: Someone close to Alta´r was wrongfully accused of the execution of Alta´r's son and when Alta´r confronts Abbas, he tosses him the head of that person — Malik a-Sayf.
The fourth one: Alta´r came back to Masyaf for revenge over two decades later, by which time Alta´r was eighty-two years old, in a tattered brown robe and now only able to jog for a few meters before he involuntarily has to stop to cough...
The sixth one: Where the now-92-year-old Alta´r, having dispersed the Order around the world and given Niccolo Polo both the Codex and the memory-encoded keys in the fifth memory, shares one last goodbye and hug with his own aged son Darim before sealing himself and the Apple inside his library underneath Masyaf, essentially burying himself alive... and then the game forces you to walk Alta´r through his last moments. The bits of dialogue Alta´r hears as he walks through the library especially hit home:
Maria: Get rid of that thing!
Alta´r: This is my duty, Maria!
Alta´r: (a few seconds later, sad and confused) Maria? Where... where are you? Where is she?!
Darim: Gone, father. You do not remember? She's gone!
The sixth one: There is a chair in the middle of the room. When the player is done with everything else in there, the game gently directs the player towards it. The action command for it is "Sit a moment and rest..." After a short while, the player realises that Alta´r isn't getting up, ever again.
In the fourth memory, as eighty-two year-old Alta´r makes his way to Abbas, you can see a glowing translucent figure sometimes around corners and in the crowds, but it vanishes before you can get close. It's Maria Thorpe. Alta´r has returned to the place where the woman he loves died, and he can not. Stop. Seeing her. Everywhere. Switch on eagle vision during those moments Alta´r will see flashbacks of his and hers visit 20 years prior. They are their last memories together still haunting him.
Revelations actually has quite a few Tearjerker moments in the final third of the game:
The third Alta´r memory with Maria's death and Alta´r's exile Made all the worst if you read the novel The Secret Crusade, where the Alta´r memories are taken from. In the novel, you learn what happened to Malik, especially concerning what happened in Masyaf while Alta´r had been in the East dealing with the Mongols with Maria and Darim. Malik is accused of Sef's murder and is imprisoned, thus allowing Abbas to built a council of Assassins in which he leads to take over the Order. When Alta´r returns he confronts Malik in prison and learns the truth, frees him, and goes to confront Abbas. Right before he hands Abbas the Apple, Alta´r is thrown a burlap sack. It's Malik's head...
Ezio discovering Yusuf's body in the Assassin's Den and quietly saying something akin to "You have earned the rest, Brother."
The final sequence where Ezio discovers Alta´r's final resting place and the final Masyaf key. "Sit a moment and rest..." indeed...
Abbas's death. Just seeing him finally concede that Alta´r is the better Assassin, and finally accepting that Alta´r told the truth about his father's suicide, which is pretty much the only thing that made him take over the Brotherhood and drive Alta´r into exile. Especially when he wonders if he can see his father again and ask him if Alta´r was right.
The fact that Ezio lives his whole life in search for answers after his family's execution, and in the last moments of Revelations, it's revealed to him that answers will never be his. His purpose is simply as a messenger, to pass the information on; he himself won't be granted the knowledge. It's this moment where he chooses to put the Assassin life behind him.
Revelations was emotionally heavy through and through. From this being Ezio's last game to the deaths of beloved characters, what really makes the Tear Jerker hit critical was the end. As in, the end of the world. Here we have the first civilization, and then that solar flare. To all of those people, their whole world came crashing down on them, and the camera seems to love to focus on a young mother and her baby, right up until a wave of fire engulfs them... Combined with the music, one can't help but feel goosebumps after watching it. And not just the first time either.
The Recruit system may have been introduced in the previous game, but losing one does not make it any easier.
Some of the NPC voices that respond to you suddenly shoving them and toppling them over are rather hurt.
"I-is there a reason for this abuse?"
Tarik. This is the first time ever in Assassin's Creed where you wind up killing someone who was actually an ally, and the game makes no secret of just how big of a mistake you just made.
The backstories of some of the multiplayer characters. For instance, Vali cel Tradat, a.k.a. The Sentinel, has this death speech:
Once your Creed was as vital to me as air and water... but when the Turks marched into Wallachia, and you Assassins did nothing to stop it, how could I continue to believe? If a man's philosophy does not let him protect his people, his home, and his family... what good can it do for the world?