Myelin proteolipid protein (PLP) 139-151 is an immunodominant peptide that induces experimental autoimmune encephalomyelitis (EAE) in H-2(s) SJL/J mice. While PLP 139-151-specific TCR transgenic (tg) 4E3 mice develop fulminant spontaneous disease on the susceptible SJL/J background, spontaneous EAE is dramatically reduced on the H-2(s) congenic B10.S background. On this resistant background, we observed a high frequency of positively selected tg CD4-CD8- (DN) thymocytes and peripheral DN tg T cells. Splenic DN tg T cells responded to anti-CD3 stimulation similarly to CD4+ cells, but proliferative and cytokine responses to PLP 139-151 were blunted, implying that CD4 co-receptor down-regulation modulated T cell responses to the self-antigen in vitro. Adoptive transfer of tg DN CD3hi cells into RAG-deficient wild-type (WT) recipients induced EAE less efficiently than transfer of CD4+ T tg cells indicating the blunted responses of DN tg T cells to self-antigen in vivo. The frequency of tg DN T cells was irrespective of thymic expression of the autoantigen. These data implicate that down-regulation of CD4 co-receptor in the thymus, which is independent from the expression of thymic autoantigen, results in a blunted response to the autoantigen in the periphery and limits the incidence of spontaneous autoimmunity in genetically resistant mice bearing a large autoreactive tg T cell repertoire.