6. Joker - Batman: Arkham City
Sure you're not going to be crying your eyes out Harley Quinn-style over 'Mr. J's death, but there's no denying it was devastating on multiple levels as an otherwise-successful Batman was dealt a monumental blow; being forced to watch his greatest nemesis slowly die in front of him. The great thing is Joker does it to himself, stabbing Batman and causing a phial of Mr. Freeze's antidote to spill out instead of cure him - leading to the Dark Knight repeating that despite everything Joker has done, he would've still saved him. Joker's last words are "That is... pretty funny!" before laughing himself to death, his lifeless body then carried out for all to see. https://www.youtube.com/watch?v=DMYF3xNd4HM Being that it's so unexpected - hardly any Bat-stories actually kill Joker off for good - it immediately made you question how Rocksteady were going to continue without the character if they do more games, as well as wondering what this all meant for Bruce Wayne as a man devoted to protecting everyone in Gotham. Luckily
Joker's role in Arkham isn't quite over thanks to the events of the ongoing Arkham Knight comic, but as for the immediacy of how impactful this scene was, it's rarely been beaten.