Introduction of alloantigens into the AC induces a form of immune tolerance known as ACAID, which induces antigen-specific CD8+ Tregs, contributing to ocular immune privilege by down-regulating immune responses. Recent evidence suggests IFN-γ is needed for the suppressive function of CD8+ ACAID Tregs. This study tested the hypothesis that IFN-γ is needed for alloantigen-specific ACAID CD8+ Tregs to execute their suppressive function but is not required for the establishment of ACAID CD8+ Tregs. To address this hypothesis, ACAID was induced by injecting BALB/c spleen cells into the AC of WT C57BL/6 mice, IFN-γ(-/-) C57BL/6 mice, or anti-IFN-γ-treated WT C57BL/6 mice. LAT assays using C57BL/6 APCs as stimulators, CD4+ T cells from C57BL/6 mice previously immunized toward BALB/c alloantigens as effector cells, and IFN-γ-competent, IFN-γ(-/-), or IFN-γR(-/-) CD8+ Tregs were used to evaluate the suppressive function of CD8+ ACAID Tregs in response to IFN-γ. IFN-γ(-/-) mice or mice treated with anti-IFN-γ antibody prior to AC injection of alloantigen failed to develop ACAID. The suppressive function of IFN-γ(-/-) ACAID CD8+ Tregs was restored through the administration of exogenous IFN-γ. This suppressive responsiveness toward IFN-γ was CD8+ Treg-intrinsic, as CD8+ Tregs from IFN-γR(-/-) mice, which were primed in the AC with alloantigens, were not able to suppress alloantigen-specific DTH responses. These results indicate that IFN-γ is not needed for the induction of CD8+ ACAID Tregs but is required for ACAID Tregs to exert the suppression of allospecific DTH responses.