We show that the mouse macrophage-restricted F4/80 protein is not required for the development and distribution of tissue macrophages but is involved in the generation of antigen-specific efferent regulatory T (T reg) cells that suppress antigen-specific immunity. In the in vivo anterior chamber (a.c.)–associated immune deviation (ACAID) model of peripheral tolerance, a.c. inoculation of antigen into F4/80^−/− mice was unable to induce efferent T reg cells and suppress delayed-type hypersensitivity (DTH) responses. Moreover, the use of anti-F4/80 mAb and F4/80^−/− APCs in an in vitro ACAID model showed that all APC cells in the culture must be able to express F4/80 protein if efferent T reg cells were to be generated. In a low-dose oral tolerance model, WT but not F4/80^−/− mice generated an efferent CD8⁺ T reg cell population that suppressed an antigen-specific DTH response. Peripheral tolerance was restored in F4/80^−/− mice by adoptive transfer of F4/80⁺ APCs in both peripheral tolerance models, indicating a central role for the F4/80 molecule in the generation of efferent CD8⁺ T reg cells.