Forkhead box P3 (FOXP3)⁺CD4⁺CD25⁺ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-β (TGF-β) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4⁺ T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-β (CBFβ) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4⁺ T cells into Foxp3⁺ iT reg cells was significantly decreased in adoptively transferred Cbfb^F/F CD4-cre naive T cells into Rag2^−/− mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and Cbfb^F/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4⁺ CD25^high CD127⁻ T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-β mRNA compared with CD4⁺CD25⁻ cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-β–induced Foxp3 expression in iT reg cell differentiation and function.