The class IV semaphorin Sema4A provides a costimulatory signal to T cells. To investigate the possible developmental and regulatory roles of Sema4A in vivo, we generated Sema4A-deficient mice. Although Sema4A-deficient mice develop normally, DCs and T cells from knockout mice display poor allostimulatory activities and T helper cell (Th) differentiation, respectively. Interestingly, in addition to its expression on DCs, Sema4A is upregulated on Th1-differentiating cells, and it is necessary for in vitro Th1 differentiation and T-bet expression. Consequently, in vivo antigen-specific T cell priming and antibody responses against T cell-dependent antigens are impaired in the mutant mice. Additionally, Sema4A-deficient mice exhibit defective Th1 responses. Furthermore, reconstitution studies with antigen-pulsed DCs reveal that DC-derived Sema4A is important for T cell priming, while T cell-derived Sema4A is involved in developing Th1 responses. Collectively, these results indicate a nonredundant role of Sema4A not only in T cell priming, but also in the regulation of Th1/Th2 responses.