The transcription factor Foxp3 has an indispensable role in establishing stable transcriptional and functional programs of regulatory T cells (T_reg cells). Loss of Foxp3 expression in mature T_reg cells results in a failure of suppressor function, yet the molecular mechanisms that ensure steady, heritable Foxp3 expression in the T_reg cell lineage remain unknown. Using T_reg cell–specific gene targeting, we found that complexes of the transcription factors Runx and CBFβ were required for maintenance of Foxp3 mRNA and protein expression in T_reg cells. Consequently, mice lacking CBFβb exclusively in the T_reg cell lineage had a moderate lymphoproliferative syndrome. Thus, Runx-CBFβ complexes maintain stable high expression of Foxp3 and serve as an essential determinant of T_reg cell lineage stability.