Introduction of antigens into the anterior chamber (AC) of the eye generates a specific systemic form of tolerance that is termed AC-associated immune deviation (ACAID). Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human CNS demyelinating diseases, including multiple sclerosis (MS) and acute disseminated encephalomyelitis. We investigated whether the encephalitogenic antigens myelin oligodendrocyte glycoprotein (MOG35–55) or myelin basic protein (MBP) induce ACAID in the EAE-prone C57BL/6 mice. We hypothesized that injection of MOG35–55/MBP induces antigen-specific tolerance whether via the AC route, the adoptive transfer of in vitro-generated MOG35–55-specific/MBP-specific ACAID antigen presenting cells (APCs), or the adoptive transfer of MOG35–55-specific/MBP-specific ACAID T regulatory cells (Tregs). ACAID is characterized by the specific impairment of delayed-type hypersensitivity (DTH) responses. Thus, DTH assays were used to test for ACAID following the AC injection of MOG35–55/MBP, or the intravenous injection of MOG35–55-specific/MBP-specific ACAID APCs. The functional local adoptive transfer (LAT) assays were used to examine the putative regulatory functions of in vitro generated MOG35–55-specific/MBP-specific Tregs. This report is the first to demonstrate the in vivo and in vitro induction of MOG35–55-specific/MBP-specific ACAID-mediated tolerance in C57BL/6 mice. These findings highlight the need for novel immunotherapeutic strategies for MS and optic neuritis.