Differentiation of naive CD4 T cells into cytokine-secreting effector Th1 and Th2 cells is influenced by several factors. We have previously reported that the affinity of antigen for TCR and antigen dose can influence the differentiation of Th1 and Th2 cells. Several in vitro and in vivo models have demonstrated a role for the costimulatory molecules, B7-1 (CD80) and B7-2 (CD86), in the generation of distinct effector T cell responses. To determine whether the strength of TCR signaling controls the involvement of CD28 costimulation in selective CD4 T cell differentiation, naive CD4 T cells bearing a transgenic TCR are primed by a weak or strong TCR signal (signal 1) in the presence or absence of B7 costimulatory molecules (signal 2). In this system, IL-4-producing Th2 cells are generated by priming with a weak but not a strong TCR signal. Th2 cell differentiation is dependent on CD28/B7 interactions in that disruption of CD28/B7 interactions inhibits the priming of Th2 cells and cross-linking CD28 with anti-CD28 antibody augments the priming of Th2 cells. In contrast, however, IL-4-producing Th2 cells cannot be generated by priming with a strong TCR signal even in the presence of strong costimulation or high doses of IL-2. Thus, our results suggest that naive CD4 T cells are receptive to CD28-dependent IL-4 production only if they receive a weak TCR signal.