Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (iDC) is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)-β and interleukin (IL)-10 double-positive CD4+ T cells within 1 week of autologous DC/T cell co-cultures. In iDC/T cell cultures, where antigen-specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF-β and IL-10 secreted by CD4+CD25−FOXP3− T cells. In addition, the suppressive capacity of CD4+ T cells conditioned by iDC was transferable to already primed antigen-specific CD8+ T cell cultures. In contrast, addition of CD4+ T cells conditioned by mDC to primed antigen-specific CD8+ T cells resulted in enhanced CD8+ T cell responses, notwithstanding the presence of TGF-β+/IL-10+ T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine-secreting regulatory T cells. We show that iDC-conditioned CD4+ T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4+ T cells. Furthermore, TGF-β+/IL-10+ T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.