The transcription factor Foxp3 is required for the development of regulatory T cells (T_reg cell). Here we report that induced ablation of a loxP-flanked Foxp3 allele in mature T_reg cells resulted in the loss of their suppressive function in vivo and acquisition of the ability to produce interleukin 2 and T helper type 1 cytokines. Furthermore, after adoptive transfer in the absence of functional T_reg cells into lymphopenic hosts, T_reg cells with deletion of Foxp3 proliferated and were predominant among tissue-infiltrating T cells. In agreement with those results, we found deregulation of Foxp3 target gene expression after Foxp3 deletion. Thus, continued Foxp3 expression in mature T_reg cells is needed to maintain the transcriptional and functional program established during T_reg cell development.