Foxp3+ CD25+ CD4+ regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-β-and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8+ DEC-205/CD205+ DCs, but not the major fraction of CD8− DC inhibitory receptor-2 (DCIR2)+ DCs, induce functional Foxp3+ Treg from Foxp3− precursors in the presence of low doses of Ag but without added TGF-β. CD8+ CD205+ DCs preferentially express TGF-β, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-β. In contrast, CD8− DCIR2+ DCs better induce Foxp3+ Treg when exogenous TGF-β is supplied. In vivo, CD8+ CD205+ DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11. 10 RAG−/− Foxp3− CD4+ T cells, whereas the CD8− DCIR2+ DCs better stimulate natural Foxp3+ Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3+ Treg, in part through the endogenous formation of TGF-β. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3+ Treg for treatment of autoimmune diseases, transplant rejection, and allergy.