We report that disruption of CD154 in nonobese diabetic (NOD) mice abrogates the helper function of CD4⁺CD25⁻ T cells without impairing the regulatory activity of CD4⁺CD25⁺ T cells. Whereas CD4⁺ T cells from NOD mice enhanced a diabetogenic CD8⁺ T cell response in monoclonal TCR-transgenic NOD mice, CD4⁺ T cells from NOD.CD154^−/− mice actively suppressed it. Suppression was mediated by regulatory CD4⁺CD25⁺ T cells capable of inhibiting CD8⁺ T cell responses induced by peptide-pulsed dendritic cells (DCs), but not peptide/MHC monomers. It involved inhibition of DC maturation, did not occur in the presence of CD154⁺ T-helper cells, and could be inhibited by activation of DCs with LPS, CpG DNA, or an agonistic anti-CD40 mAb. Thus, in at least some genetic backgrounds, CD154-CD40 interactions and innate stimuli release immature DCs from suppression by CD4⁺CD25⁺ T cells.