Recent studies have highlighted the importance of peripheral induction of Foxp3-expressing regulatory T cells (Tregs) in the dominant control of immunological tolerance. However, Foxp3⁺ Treg differentiation from naïve CD4⁺ T cells occurs only under selective conditions, whereas the classical T helper (Th) 1 and 2 effector development often dominate T cell immune responses to antigen stimulation in the periphery. The reason for such disparity remains poorly understood. Here we report that Th1/Th2-polarizing cytokines can potently inhibit Foxp3⁺ Treg differentiation from naïve CD4⁺ precursors induced by TGF-β. Furthermore, antigen receptor-primed CD4⁺ T cells are resistant to Treg induction because of autocrine production of IFNγ and/or IL-4, whereas neutralizing IFNγ and IL-4 not only can potentiate TGF-β-mediated Foxp3 induction in vitro but can also enhance antigen-specific Foxp3⁺ Treg differentiation in vivo. Mechanistically, inhibition of Foxp3⁺ Treg development by Th1/Th2-polarizing cytokines involves the activation of Th1/Th2 lineage transcription factors T-bet and GATA-3 through the canonical Stat1-, Stat4-, and Stat6-dependent pathways. Using IFNγ and IL-4 knockouts and retrovirus-mediated transduction of T-bet and GATA-3, we further demonstrate that enforced expression of the Th1/Th2 lineage-specific transcription factors is sufficient to block Foxp3 induction and Treg differentiation independent of the polarizing/effector cytokines. Thus, our study has unraveled a previously unrecognized mechanism of negative cross-regulation of Foxp3⁺ Treg fate choice by Th1/Th2 lineage activities. In addition, these findings also provide an attainable explanation for the general paucity of antigen-triggered de novo generation of Foxp3⁺ Tregs in the periphery.