Induced regulatory T cells (iT(reg) cells) can be generated by peripheral dendritic cells (DCs) that mediate T cell unresponsiveness to rechallenge with antigen. The molecular factors required for the function of such iT(reg) cells remain unknown. We report a critical role for the transcription cofactor homeodomain-only protein (Hop; also known as Hopx) in iT(reg) cells to mediate T cell unresponsiveness in vivo. Hopx-sufficient iT(reg) cells downregulated expression of the transcription factor AP-1 complex and suppressed other T cells. In the absence of Hopx, iT(reg) cells had high expression of the AP-1 complex, proliferated and failed to mediate T cell unresponsiveness to rechallenge with antigen. Thus, Hopx is required for the function of T(reg) cells induced by DCs and the promotion of DC-mediated T cell unresponsiveness in vivo.