To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the adaptive immune response following mucosal immunization is required. CD11c(high) conventional dendritic cells (cDCs) have been shown to be necessary for activation of naive CD8(+) T cells in vivo, but the role of cDCs in CD4(+) T cell activation is still unclear, especially at mucosal surfaces. The activation of naive Ag-specific CD4(+) T cells and the generation of Abs following mucosal administration of Ag with or without the potent mucosal adjuvant cholera toxin were therefore analyzed in mice depleted of CD11c(high) cDCs. Our results show that cDCs are absolutely required for activation of CD4(+) T cells after oral and nasal immunization. Ag-specific IgG titers in serum, as well as Ag-specific intestinal IgA, were completely abrogated after feeding mice OVA and cholera toxin. However, giving a very high dose of Ag, 30-fold more than required to detect T cell proliferation, to cDC-ablated mice resulted in proliferation of Ag-specific CD4(+) T cells. This proliferation was not inhibited by additional depletion of plasmacytoid DCs or in cDC-depleted mice whose B cells were MHC-II deficient. This study therefore demonstrates that cDCs are required for successful mucosal immunization, unless a very high dose of Ag is administered.