Little is known about the molecules that control the development and function of CD4⁺CD25⁺ Tregs. Recently, it was shown that the transcription factor FOXP3 is necessary and sufficient for the generation of CD4⁺CD25⁺ Tregs in mice. We investigated the capacity of FOXP3 to drive the generation of suppressive CD4⁺CD25⁺ Tregs in humans. Surprisingly, although ectopic expression of FOXP3 in human CD4⁺ T cells resulted in induction of hyporesponsiveness and suppression of IL-2 production, it did not lead to acquisition of significant suppressor activity in vitro. Similarly, ectopic expression of FOXP3Δ2, an isoform found in human CD4⁺CD25⁺ Tregs that lacks exon 2, also failed to induce the development of suppressor T cells. Moreover, when FOXP3 and FOXP3Δ2 were simultaneously overexpressed, although the expression of several Treg-associated cell surface markers was significantly increased, only a modest suppressive activity was induced. These data indicate that in humans, overexpression of FOXP3 alone or together with FOXP3Δ2 is not an effective method to generate potent suppressor T cells in vitro and suggest that factors in addition to FOXP3 are required during the process of activation and/or differentiation for the development of bona fide Tregs.