Hmmmm, this one is a toughie. There are quite a few that I've really enjoyed - Castiel, Death, Lucifer, Meg, Ruby, Trickster/Gabriel but my favourite has to be:
Technically, it's not Sam's very first appearance, but it is the first time we see Jared on screen.
You coming or what?
Do I have to?
Yes! It'll be fun.
SAM comes into the room.
And where's your costume?
You know how I feel about Halloween.
I admit a lot of my love for this scene is just how young and cute Jared looks. Shallow, I know but that's just how I roll. :-)