It happens right before Dean gives Jo the “last night on earth” speech. Dean and Sam are busy discussing how to kill the devil, and Dean’s attention is distracted by Jo. He looks over at her, Ellen and Cas, and frowns at what he sees:

Jo is clearly dominating the shot, but what you might not notice on first glance is how she’s leaning in towards Cas, her hands near his arm on the table. Her gaze is clearly on him and not her mom; whatever she has said, she has said to him alone. And she has just let loose a particularly flirtatious sounding laugh. A giggle, even.
The dialogue that caught Dean’s attention is hard to hear apart from her laughter, but what I think Jo says to Cas there is, “Cas, (*giggle*), stop it. Drink up.” It’s hard to make out because she’s using that cute-coy laughing tone that young women do when they’re flirting (she uses it when she’s pretending to be Dean’s girlfriend in the H.H. Holmes episode, and hell, I’ve used it plenty in my own life).
Then, as the camera stays on her, we see her push up from the table and declare, “I’m gonna go get another beer.” Her gaze lingers on Cas, who returns it (albeit more briefly):

When the camera switches back to Dean, he’s making this sour face, which he holds for several seconds, as he assesses the situation before him:

Sam has his eyes on the drinking contest before him and says to Dean, “Talk about stupid ideas”, and at first I thought Sam was talking about the drinking contest. But once I noticed what appears to be Jo flirting with Cas, now I’m not so sure.
In any event, Dean, whose eyes never leave the table where Ellen and Cas sit, murmurs “Good God; true that”, and gets up. He walks over to wear Jo is digging through the fridge for a beer, then stops and stands way too close to her, so much so that when she turns around she’s visibly startled when she turns around and greets him.
Then Dean turns around, sidles beside her, and clearly looks over at the table where Ellen and Cas are sitting before he launches into “Dangerous mission tomorrow”, which is the opening of his “last night on earth” speech:

But even as he says “dangerous mission tomorrow,” his eyes never leave Cas.

I say he’s looking at Cas, specifically, because Dean’s gaze is clearly in the middle distance in both these shots. In this configuration of the scene, Ellen is sitting close to Dean, only a few feet away with her back toward him, while Cas is facing him but further away. The kitchen is small enough that if Dean were looking at Ellen, he’d appear more as if he were looking down, instead of out.
You can see the difference when he continues with his speech, “I guess it’s time to eat, drink—”

His eyes are much closer; they’ve landed on Ellen — and for good reason, because he’s basically about to proposition her daughter, and Dean’s worried Ellen might overhear; he doesn’t continue with the proposition, “and, y’know, make merry,” until he’s sure Ellen hasn’t overheard.
Anyway, so what’s the point of this second-by-second replay?
I used to think that this scene was fairly out of character for Dean. Canonically, he’s never looked at Jo as anything other than a little sister, and the fact that he suddenly wants to sleep with her now can only really be explained away by Dean’s need for physical comfort in the face of certain death tomorrow.
But I’d also never noticed before what appears to be Jo flirting with Dean’s best friend, teasing him, leaning in close, trying to get him drunk, and so on. Maybe Jo’s actually attempting to work a little “last night on earth” mojo of her own, which Dean sees and attempts to hurl himself in front of before it can even occur.
But why? Is it just more comfort-seeking on his part (as in, “if someone’s getting laid tonight, it had better be me”)? Or is the answer in the way Dean’s gaze lingers on Cas specifically, even as he’s propositioning Jo? That is to say, does Dean proposition Jo in front of Cas out of jealousy—not of Cas, but of Jo?
I dunno. Just some food for thought.
I actually noticed this too, but didn’t say anything in fear that I would just be “reaching” for it.