In the course of autoimmune CNS inflammation, inflammatory infiltrates form characteristic perivascular lymphocyte cuffs by mechanisms that are not yet well understood. Here, intravital two-photon imaging of the brain in anesthetized mice, with experimental autoimmune encephalomyelitis, revealed the highly dynamic nature of perivascular immune cells, refuting suggestions that vessel cuffs are the result of limited lymphocyte motility in the CNS. On the contrary, vessel-associated lymphocyte motility is an actively promoted mechanism which can be blocked by CXCR4 antagonism. In vivo interference with CXCR4 in experimental autoimmune encephalomyelitis disrupted dynamic vessel cuffs and resulted in tissue-invasive migration. CXCR4-mediated perivascular lymphocyte movement along CNS vessels was a key feature of CD4⁺ T cell subsets in contrast to random motility of CD8⁺ T cells, indicating a dominant role of the perivascular area primarily for CD4⁺ T cells. Our results visualize dynamic T cell motility in the CNS and demonstrate differential CXCR4-mediated compartmentalization of CD4⁺ T-cell motility within the healthy and diseased CNS.