Most Foxp3 regulatory T (T) cells develop in the thymus as a functionally mature T cell subpopulation specialized for immune suppression. Their cell fate appears to be determined before Foxp3 expression; yet molecular events that prime Foxp3 T precursor cells are largely obscure. We found that T cell-specific super-enhancers (T-SEs), which were associated with Foxp3 and other T cell signature genes, began to be activated in T precursor cells. T cell-specific deficiency of the genome organizer Satb1 impaired T-SE activation and the subsequent expression of T signature genes, causing severe autoimmunity due to T cell deficiency. These results suggest that Satb1-dependent T-SE activation is crucial for T cell lineage specification in the thymus and that its perturbation is causative of autoimmune and other immunological diseases.