Naturally occurring CD4⁺CD25⁺ regulatory T (T_R) cells play crucial roles in normal immunohomeostasis. CD4⁺CD25⁺ T_R cells exhibit a number of interesting in vitro properties including a ‘default state’ of profound anergy refractory to conventional T cell stimuli. We investigated the in vitro activation requirements of CD4⁺CD25⁺ T_R cells using bone marrow-derived DC, which as professional antigen presenting cells (APC) can support the activation of normal naïve T cells. Comparison of different APC types revealed that LPS-matured DC were by far the most effective at breaking CD4⁺CD25⁺ T_R cell anergy and triggering proliferation, and importantly their IL-2 production. Examination of Foxp3, a key control gene for CD4⁺CD25⁺ T_R cells, showed this to be stably expressed even during active proliferation. Although CD4⁺CD25⁺ T_R cell proliferation was equivalent to that of CD25⁻ cells their IL-2 production was considerably less. Use of IL-2^−/− mice demonstrated that the DC stimulatory ability was not dependent on IL-2 production; nor did IL-15 appear crucial but was, at least in part, related to costimulation. DC also blocked normal CD4⁺CD25⁺ T_R cell-mediated suppression partially via IL-6 secretion. DC therefore possess novel mechanisms to control the suppressive ability, expansion and/or differentiation of CD4⁺CD25⁺ T_R cells in vivo.