CD40 ligand (CD40L) gene-disrupted (CD40L−/−) mice were employed to examine the role of costimulatory signals via CD40L-CD40 interactions in mucosally induced tolerance. CD40L−/− and control (CD40L+/+) mice of the same C57BL/6× 129/J background were immunized orally with 25 mg of OVA before systemic challenge with OVA in CFA. While CD40L+/+ mice showed reductions in Ag-specific T cell responses including delayed-type hypersensitivity (DTH) and proliferative responses, CD40L−/− mice underwent normal T cell responses. Further, cytokine analysis of splenic CD4+ T cells showed that both Th1-type (eg, IFN-γ and IL-2) and Th2-type (eg, IL-4, IL-5, IL-6, and IL-10) responses were maintained in CD40L−/− mice orally immunized with OVA, whereas these cytokine responses in CD40L+/+ mice were significantly reduced. In addition, splenic CD4+ T cells from CD40L−/− mice orally immunized with OVA provided B cell help in Ag-specific Ab-forming cells when the cells were cultured with naive B cells in the presence of Ag and CD40L-transfected cell lines. In contrast, an identical culture condition containing splenic CD4+ T cells from orally tolerized CD40L+/+ mice did not exhibit helper activity. Taken together, these findings indicate that CD40L and CD40 interactions are essential for the induction of systemic T cell unresponsiveness to orally administered Ag.