Uncle Ben: You're a lot like your father. You really are, Peter, and that's a good thing. But your father lived by a philosophy, a principle really. He believed that...that if you could do good things for other people, you had a moral obligation to do those things! That's what at stake here. Not a choice, responsibility. Peter: That is nice. That's really...that's great. That's all well and good, so where is he? Uncle Ben: What? Peter: Where is he? Where's my dad? He didn't think it was his responsibility to be here and tell me this himself? Uncle Ben: Oh, come on! How dare you? Peter: How dare I? How dare you?!
There's also his posthumous voicemail to Peter as well.
Also the fact that after Peter walks away, Ben went looking for him to help with whatever grief he's feeling. Though May thinks they should give him some time alone, but Ben doesn't want things to be left unsaid.
Plus, Ben must feel lousy for bringing up Peter's father, despite knowing it's a touchy subject for his nephew.
Gwen showing up at Peter's house after her father's funeral and desperately asking him why he wasn't there.
Everything Aunt May goes through, her family slowly being ripped apart first with the Parkers disappearing, then her husband being shot, and her nephew that she raised coming home late at night covered in cuts and bruises. And then at the end of film, he comes home covered in blood and she just holds him tightly.
Flash's words after Peter slams him against the locker explain a lot about the character in very little time.
Flash: It feels better right? Look, your uncle died. I'm sorry. I get it. I'm sorry.
Captain Stacy's death. He starts off as a rather unsympathetic character and grows on you as you see his reasoning, and that he's not just a one-note take-the-vigilante-off-my-streets copper, until at last he gets the chance to stop Peter, and lets him go. And saves his life. And then gets killed. Also, Stacy asking Peter to break up with Gwen so that she's not put in danger by the enemies he'll make as Spider-Man.
Likewise, the fate of Dr. Connors: pushed into taking the serum when his financial backer shuts him down, it's strongly implied that he had little control over what his lizard-self wanted and did. He is horrified when he regains full humanity and sees what he's done. (Yes, he was responsible and deserved punishment, since the choice to take the serum was ultimately his — but sad, even so.)
What's even worse is he didn't take the serum just For Science!. He took it because they were about to test it on wounded veterans and he was trying to minimize casualties if the serum went wrong. (And, of course, it did.)