Human regulatory T cells (T_R) cells have potential for the treatment of a variety of immune mediated diseases but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human T_R cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimulation with a superagonistic anti-CD28 antibody (clone 9.3) and IL-2 partially reversed the proliferative defect, and this correlated with reversal of the defective calcium mobilization in these cells. Dendritic cells were effective at promoting T_R cell proliferation, and under these conditions the proliferative capacity of T_R cells was comparable to conventional CD4 lymphocytes. Blocking TGF-β activity abrogated IL-10 expression from these cells, while addition of TGF-β resulted in IL-10 production. These data demonstrate that highly purified populations of T_R cells are anergic even in the presence of high doses of IL-2. Furthermore, antigen presenting cells provide proper co-stimulation to overcome the anergic phenotype of T_R cells, and under these conditions they are highly sensitive to IL-2. In addition, these data demonstrate for the first time that TGF-β is critical to enable human T_R cells to express IL-10.