Costimulation of T cell activation involves both the B7: CD28 as well as the CD40 ligand (CD40L): CD40 pathway. To determine the importance of these pathways to in vitro and in vivo T cell activation, a direct comparison was made of the responses of TCR transgenic T cells lacking either CD28 or CD40L. In vitro, CD28−/− T cells showed a greater reduction in proliferative responses to Ag than did CD40L−/− T cells. The absence of CD28 resulted in defective Th2 responses, whereas CD40L−/− T cells were defective in Th1 development. In vivo, CD28−/− T cells failed to expand upon immunization, whereas CD40L−/− T cells could not sustain a response. These results suggest that CD28 is critical for initiating T cell responses, whereas CD40L is required for sustained Th1 responses. The different functional roles of these costimulatory pathways may explain why blocking B7: CD28 and CD40L: CD40 interactions has an additive effect in inhibiting T cell responses.