Hearing Penny has made a friend at school (Ryan Cartwright of Alphas and Bones), Leonard feels insecure about his own status with Penny. Similar scenarios have played out on many sitcoms, but this one stood out because Penny had never told Leonard she loves him. She's been overtly pressured to say so since last season, and less overtly since Season 3.
The words finally came in frustration, Penny saying, "Listen to me. You're the one I'm with. You know I love you, so will you please relax because you're driving me crazy."
After a beat, Leonard pointed out that was the first time Penny said she loved him. I didn't notice it myself, her delivery was so spontaneous. The realization took Penny aback, too, driving them both to tears, living up to six seasons in the making.