CD4⁺ regulatory T cells (T_reg cells) characterized by expression of the transcription factor Foxp3 have a pivotal role in maintaining immunological tolerance. Here we show that mice with T cell–specific deletion of both the Foxo1 and Foxo3 transcription factors (collectively called 'Foxo proteins' here) developed a fatal multifocal inflammatory disorder due in part to T_reg cell defects. Foxo proteins functioned in a T_reg cell–intrinsic manner to regulate thymic and transforming growth factor-β (TGF-β)-induced Foxp3 expression, in line with the ability of Foxo proteins to bind to Foxp3 locus and control Foxp3 promoter activity. Transcriptome analyses showed that Foxo proteins regulated the expression of additional T_reg cell–associated genes and were essential for inhibiting the acquisition of effector T cell characteristics by T_reg cells. Thus, Foxo proteins have crucial roles in specifying the T_reg cell lineage.