A crucial step in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS), is transmigration of pathogenic T cells across the blood–brain barrier. These T cells mediate inflammation and subsequent lesion formation in the CNS. However, molecular mechanisms underlying lesion distribution and formation are not well understood. We here show that genetic ablation of a single immunoregulatory molecule on T cells, B7-homolog 1 (B7-H1), causes local endothelial dysfunction and determines lesion topography in a spontaneous mouse model of CNS autoimmunity. These findings can lead to new therapeutic approaches in targeting pathogenic T cell responses in MS.