CD4+CD25+forkhead box p3 (Foxp3)+ regulatory T cells (Treg) control peripheral tolerance. Although Treg are anergic when stimulated through the TCR, mature bone marrow-derived, but not splenic, dendritic cells (DC) can induce their proliferation after TCR stimulation in the absence of IL-2. One possibility is that the DC produce proinflammatory cytokines such as IL-1 or IL-6 that function as growth factors for Treg. We have analyzed the costimulatory effects of IL-1 on the expansion of Foxp3+ Treg in vitro. When CD4+CD25+ T cells were cultured in the presence of splenic DC and IL-1, marked expansion of the Foxp3+ T cells was observed. The effects of IL-1 were mediated on CD4+CD25+Foxp3(-) T cells present in the starting population rather than on the DC or on the CD4+CD25+Foxp3+ T cells. In contrast, stimulation of CD4+CD25+ T cells with plate-bound anti-CD3 and IL-1 in the absence of DC resulted in the outgrowth of a CD4+CD25+Foxp3(-) T cell population composed of NKT cells and non-NKT, IL-17-producing cells. Foxp3+ Treg purified from mice expressing the reporter gene enhanced GFP in the Foxp3 locus failed to proliferate when costimulated with IL-1. These findings have important implications for the design of protocols for the expansion of CD4+CD25+ T cells for cellular biotherapy.