Systemic immunological tolerance to Ag encountered in the eye restricts the formation of potentially damaging immune responses that would otherwise be initiated at other anatomical locations. We previously demonstrated that tolerance to Ag administered via the anterior chamber (AC) of the eye required Fas ligand-mediated apoptotic death of inflammatory cells that enter the eye in response to the antigenic challenge. Moreover, the systemic tolerance induced after AC injection of Ag was mediated by CD8+ regulatory T cells. This study examined the mechanism by which these CD8+ regulatory T cells mediate tolerance after AC injection of Ag. AC injection of Ag did not prime CD4+ T cells and led to increased TRAIL expression by splenic CD8+ T cells. Unlike wild-type mice, Trail-/- or Dr5 -/- mice did not develop tolerance to Ag injected into the eye, even though responding lymphocytes underwent apoptosis in the AC of the eyes of these mice. CD8+ T cells from Trail-/- mice that were first injected via the AC with Ag were unable to transfer tolerance to naive recipient wild-type mice, but CD8+ T cells from AC-injected wild-type or Dr5-/- mice could transfer tolerance. Importantly, the transferred wild-type (Trail+/+) CD8+ T cells were also able to decrease the number of infiltrating inflammatory cells into the eye; however, Trail-/- CD8+ T cells were unable to limit the inflammatory cell ingress. Together, our data suggest that "helpless" CD8+ regulatory T cells generated after AC injection of Ag enforce systemic tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye.