Vα14 NKT cells produce large amounts of IFN-γ and IL-4 upon recognition of their specific ligand α-galactosylceramide (α-GalCer) by their invariant TCR. We show here that NKT cells constitutively express CD28, and that blockade of CD28-CD80/CD86 interactions by anti-CD80 and anti-CD86 mAbs inhibits the α-GalCer-induced IFN-γ and IL-4 production by splenic Vα14 NKT cells. On the other, the blockade of CD40-CD154 interactions by anti-CD154 mAb inhibited α-GalCer-induced IFN-γ production, but not IL-4 production. Consistent with these findings, CD28-deficient mice showed impaired IFN-γ and IL-4 production in response to α-GalCer stimulation in vitro and in vivo, whereas production of IFN-γ but not IL-4 was impaired in CD40-deficient mice. Moreover, α-GalCer-induced Th1-type responses, represented by enhanced cytotoxic activity of splenic or hepatic mononuclear cells and antimetastatic effect, were impaired in both CD28-deficient mice and CD40-deficient mice. In contrast, α-GalCer-induced Th2-type responses, represented by serum IgE and IgG1 elevation, were impaired in the absence of the CD28 costimulatory pathway but not in the absence of the CD40 costimulatory pathway. These results indicate that CD28-CD80/CD86 and CD40-CD154 costimulatory pathways differentially contribute to the regulation of Th1 and Th2 functions of Vα14 NKT cells in vivo.