T cells interact with dendritic cells (DCs) for periods lasting from minutes to hours. However, a causal link between the duration of this interaction and the efficiency of T cell activation has not been established in vivo. Employing intravital two-photon imaging, we manipulated T cell-DC interactions in real time and found that the first T cell-DC encounter often resulted in a long-lived interaction. Moreover, the cessation of T cell receptor-major histocompatibility complex signals promoted cellular dissociation, suggesting that antigen availability on DCs regulates contact duration. Finally, at least 6 hr of in vivo T cell-DC interaction were required for naive CD4⁺ T cells to undergo clonal expansion. These results establish the importance of prolonged T cell-DC interactions for efficient CD4⁺ T cell activation in vivo.