The role of CTLA-4 in regulatory T cell (Treg) function is not well understood. We have examined the role of CTLA-4 and its relationship with the transcription factor FoxP3 using a model of Treg induction in human peripheral blood. Activation of human CD4(+)CD25(-) T cells resulted in the appearance of a de novo population of FoxP3-expressing cells within 48 h. These cells expressed high levels of CTLA-4 and cell sorting on expression of CTLA-4 strongly enriched for FoxP3(+)-expressing cells with suppressive function. Culture in IL-2 alone also generated cells with suppressive capacity that also correlated with the appearance of CTLA-4. To directly test the role of CTLA-4, we transfected resting human T cells with CTLA-4 and found that this method conferred suppression, similar to that of natural Tregs, even though these cells did not express FoxP3. Furthermore, transfection of FoxP3 did not induce CTLA-4 and these cells were not suppressive. By separating the expression of CTLA-4 and FoxP3, our data show that FoxP3 expression alone is insufficient to up-regulate CTLA-4; however, activation of CD4(+)CD25(-) T cells can induce both FoxP3 and CTLA-4 in a subpopulation of T cells that are capable of suppression. These data suggest that the acquisition of suppressive behavior by activated CD4(+)CD25(-) T cells requires the expression of CTLA-4, a feature that appears to be facilitated by, but is not dependent on, expression of FoxP3.