Abstract Signal transducers and activators of transcription 5 A and B (STAT5A & B) play a critical role in mediating cellular responses following cytokine stimulation. STAT5 proteins critically signal via the formation of dimers through SH2 domain interactions. However, tetramerization of STAT5 through dimer interactions in the N-domain, has key biological roles. STAT5 tetramers control the suppressive function of regulatory T cells (Tregs), the expansion of CD8+ T cells, and the terminal differentiation of NK cells. However, the role of STAT5 tetramerization in autoimmune-mediated neuroinflammation has not been investigated. Using the STAT5 tetramer-deficient Stat5a-Stat5b N-domain double knock-in (DKI) mouse strain, in which dimer function is preserved, we found that STAT5 tetramers promote the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. The mild EAE phenotype observed in DKI mice correlates with impaired extravasation of pathogenic Th17 cells and interactions between Th17 cells and monocyte-derived cells (MDCs) in the meninges. We further demonstrated that STAT5 tetramerization mediated by GM-CSF signaling, a key EAE-associated cytokine, regulates the production of the chemokine CCL17 by MDCs. Importantly, CCL17 can partially restore the pathogenicity of DKI Th17 cells, and this is dependent on the activity of the integrin VLA-4. Thus, our study reveals a novel GM-CSF-STAT5 tetramer-CCL17 pathway in MDCs that promotes autoimmune-mediated neuroinflammation. This work was supported by NIH grant P20 GM109098 and WVU Institutional Funds to E.C.K.W.; Praespero Innovation Award Program to E.C.K.W. and V.L.; NIH grants P20 GM103434 and U54 GM104942 to G.H.; the Intramural Research Program of the National Heart, Lung, and Blood Institute, NIH to W.J.L.; the Intramural Research Program of the National Cancer Institute, NIH to V.L. NIH grants S10 OD016165, U57 GM104942, P30 GM103488, and P20 GM103434 to the WVU Flow Cytometry & Single Cell Core Facility. NIH grants U54 GM104942, P20 GM103434 and P20 GM121299 to the WVU and Marshall University Genomics Cores. Funds from WVU Cancer Institute, WVU HSC Office of Research and Graduate Education, and NIH grants P20 RR016440, P30 GM103488, P20 GM121322, U54 GM104942, and P20 GM103434 to the WVU Microscope Imaging Facility. K.L.M. was supported through The American Association of Immunologists Careers in Immunology Fellowship Program.