CD4+ CD25+ regulatory T cells (Tregs) have far-reaching immunotherapeutic applications, the realization of which will require a greater understanding of the factors influencing their function and phenotype during ex vivo manipulation. In murine models, IL-2 plays an important role in both the maintenance of a functional Treg population in vivo and the activation of suppression in vitro. We have found that IL-2 maintains optimal function of human CD4+ CD25+ Tregs in vitro and increases expression of both forkhead box protein 3, human nomenclature (FOXP3) and the distinctive markers CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor superfamily member number 18 (GITR). Although IL-2 reduced spontaneous apoptosis of Tregs, this property alone could not account for the optimal maintenance of the regulatory phenotype. The inhibition of phosphatidylinositol 3-kinase (PI3K) signaling by LY294002, a chemical inhibitor of PI3K, abolished the maintenance of maximal suppressive potency by IL-2, yet had no effect on the up-regulation of FOXP3, CD25, CTLA-4 and GITR. Other common gamma chain (gammac) cytokines-IL-4, IL-7 and IL-15-had similar properties, although IL-4 showed a unique lack of effect on the expression of FOXP3 or Treg markers despite maintaining maximal regulatory function. Taken together, our data suggest a model in which the gammac cytokines IL-2, IL-4, IL-7 and IL-15 maintain the optimal regulatory function of human CD4+ CD25+ T cells in a PI3K-dependent manner, offering new insight into the effective manipulation of Tregs ex vivo.