Regulatory T cells (T_reg cells) expressing the transcription factor Foxp3 are key in maintaining the balance of immune homeostasis. However, distinct induced T regulatory type 1 (Tr1) cells that lack Foxp3 expression also regulate T cell function, mainly by producing the immunosuppressive cytokine interleukin 10 (IL-10). However, the factors required for the induction of IL-10-producing suppressive T cells are not fully understood. Here we demonstrate that dendritic cells modified by T_reg cells induced the generation of IL-10-producing Tr1 cells. The differentiation of naive CD4⁺ T cells into IL-10-producing cells was mediated by IL-27 produced by the T_reg cell–modified dendritic cells, and transforming growth factor-β amplified the generation of induced IL-10⁺ Tr1 cells by IL-27. Thus, IL-27 and transforming growth factor-β promote the generation of IL-10-producing Tr1 cells.