Gru: Okay, well, then...maybe you could just use your imagination.
Agnes: You mean I pretend I have a mom?
Gru: Yes, right. You can do that, can't you?
Agnes:(Happily) Yeah! I do that all the time! Thanks, Gru! (She kisses Gru on the cheek and trots away, leaving Gru surprised over what she just said)
Gru sitting on the steps in the rain after learning Lucy is being relocated to Australia. Doubly hurts because this is immediately after he realizes he's in love with her. Then Agnes comes out with an umbrella and it gets a bit worse.
Gru: Remember when you said that I liked Lucy? Well...it turns out...you were right.
Gru: Yes. But, well, she is moving away. I'm never going to see her again.
(Agnes immediately stops smiling)
The flashback to young Gru trying to ask out a girl he likes, but when he tries to get her attention by tapping her on the shoulder, all the kids immediately freak out because she's got "Gruties" and flee, leaving poor Gru standing alone with the most heartbreaking expression ever on his face. The movies must enjoy making a Woobie out of Gru.
Gru's reaction to seeing a mutated minion for the first time.
Later on, we see the Minion break room nearly empty with only a small amount of Minions left. It's disconcerting considering how earlier in the movie it was filled to the brim with the little yellow guys.
Margo's first heartbreak. Gru comes back from his encounter with El Macho and Dr. Nefario and sees his daughter with a somber expression. He gets down to her level and asks what's wrong. Her answer is short, but he understands.
It's Played for Laughs, but Agnes' recting on her part for the Mother's Day Show gives some sad implications in them. If going by real life time span, Agnes was about three, when she first met Gru, before that she was an orphan. Now look at how she said her part: no emotion, no feeling in the words. Just a stoic face on a six-year-old girl. This is a heavy indication that her parents left her life before she could form a bond with either them. She already has a father-daughter relationship with Gru, but Agnes probably doesn't know what having a mom is like.
After Gru and Lucy escape the rocket and fall in the water, Gru is the first to surface. Although Lucy also surfaces ten seconds later, just seeing Gru frantically look around and yelling "Lucy! Lucy, where are you?!" is so heartbreaking to see.