Following inoculation of Ag into the anterior chamber (ac), systemic tolerance develops that is mediated in part by Ag-specific efferent CD8+ T regulatory (Tr) cells. This model of tolerance is called ac-associated immune deviation. The generation of the efferent CD8+ Tr cell in ac-associated immune deviation is dependent on IL-10-producing, CD1d-restricted, invariant Vα14+ NKT (iNKT) cells. The iNKT cell subpopulations are either CD4+ or CD4− CD8− double negative. This report identifies the subpopulation of iNKT cells that is important for induction of the efferent Tr cell. Because MHC class II−/−(class II−/−) mice generate efferent Tr cells following ac inoculation, we conclude that conventional CD4+ T cells are not needed for the development of efferent CD8+ T cells. Furthermore, Ab depletion of CD4+ cells in both wild-type mice (remove both conventional and CD4+ NKT cells) and class II−/− mice (remove CD4+ NKT cells) abrogated the generation of Tr cells. We conclude that CD4+ NKT cells, but not the class II molecule or conventional CD4+ T cells, are required for generation of efferent CD8+ Tr cells following Ag introduction into the eye. Understanding the mechanisms that lead to the generation of efferent CD8+ Tr cells may lead to novel immunotherapy for immune inflammatory diseases.