CD25+ CD4+ regulatory T cells (Tregs) are required for the maintenance of peripheral tolerance to certain self Ags. In this study, the requirements for murine Treg-suppressive activity and proliferation were examined in the context of the maturation of myeloid dendritic cells (DCs). We find that the suppressive function of Tregs is critically dependent on immature DCs and is readily reversed by the maturation of DCs induced by GM-CSF, but does not require TLR activation of either DCs or Tregs. In contrast, reversal of Treg anergy is dependent on TLR activation of DCs, and involves the potentiation of Treg responsiveness to IL-2 by cooperative effects of IL-6 and IL-1, both of which are produced by TLR-activated, mature DCs. Thus, proinflammatory cytokines produced by TLR-activated, mature DCs are required for reversal of Treg anergy, but are not required to overcome Treg suppression.