Vγ4⁺ T cells have been identified as the main IL-17-producing γδ T cell in the CNS of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The findings of this study demonstrate that Vγ4⁺ T cells are present in T cell receptor (TCR)δ^-/- mice, and furthermore these Vγ4⁺ T cells co-express TCRβ. The data reveals that Vγ4β T cells respond to IL-1β and IL-23 stimulation in the absence of TCR engagement to produce IL-17A and IL-17F and express the master transcription factor, RORγt and the integrin, MCAM.  Furthermore, Vγ4β⁺ T cells, together with conventional Vγ4δ⁺ T cells were found in the brains of mice with EAE. Vγ4β⁺T cells were also found in the brains TCRδ^-/- mice with EAE. Although, the course of EAE is somewhat reduced in TCRδ^-/- compared with wild type (WT) mice, depletion of Vγ4⁺ T cells from TCRδ^-/- as well as WT mice significantly attenuated clinical disease and weight loss in mice with EAE. Anti-Vγ4 treated mice with EAE had a significantly reduced frequency of infiltrating IL-17⁺, IFN-γ⁺ and IFN-γ⁺IL-17⁺ CD4⁺ T cells into CNS. Furthermore, in the adoptive transfer model of EAE, depletion of Vγ4⁺ cells from lymph node and spleen cells from TCRδ^-/- mice significantly impair their ability to induce EAE, with a reduction in inflammatory T cells infiltrating the CNS. Our study has identified a novel γβ T cell subset, Vγ4β T cells, which together with conventional Vγ4δ T cells, play a critical role in the pathogenesis of EAE through innate IL-17 production, necessary to enhance Th17 and Th1 activation and migration into the CNS to mediate inflammation and autoimmunity.