Abstract CD4 T cell differentiation is a process finely controlled by specific transcriptional programs leading to the acquisition of specific T helper effector functions. Up to now, the functions of Foxo3 in CD4 T cells have not been investigate. Here, we described for the first time the critical role of the transcription factors Foxo3 in Eomes expression and their involvement in pathogenic T Helper 1 differentiation. First, we showed that TCR triggering resulted in a dose-dependent upregulation of Foxo3 in CD4 T cells with an increased expression over time. We next addressed Foxo3 functions in CD4 T cells and we observed a decreased production of the Th1 related cytokines IFN-g and GM-CSF by Foxo3 KO CD4 T cells, with no impact survival, proliferation or other lineages differentiation. We then compared the transcriptome of WT and Foxo3 KO CD4 T cells and found that Foxo3 deficiency resulted in a decreased expression of genes related to the IFN- g/IFN-g response pathway and that the most downregulated gene in this pathway is Eomes. We showed, using ChIP and luciferase experiments, that Foxo3 binds and induces the expression of Eomes through direct DNA binding within the eomes locus. Using lentivirus experiments we showed overexpression of Eomes in Foxo3 KO CD4 T cell restored the proportion of IFN-g and GM-CSF producing cells, thus comforting Eomes role’s in the pathogenicity of CD4 T cells. In addition to this defect of differentiation, Foxo3-deficient mice developed a less severe Experimental Autoimmune Encephalomyelitis (EAE) as compared to WT mice, thus emphasizing the role of Foxo3 and Eomes in the development of autoimmunity. Altogether, we described here a new pathway whereby Foxo3 and Eomes drive CD4 T cells pathogenicity and neuro-inflammation.