Most Foxp3+ regulatory T (T reg) 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 reg precursor cells are largely obscure. We found that T reg cell–specific super-enhancers (T reg-SEs), which were associated with Foxp3 and other T reg cell signature genes, began to be activated in T reg precursor cells. T cell–specific deficiency of the genome organizer Satb1 impaired T reg-SE activation and the subsequent expression of T reg signature genes, causing severe autoimmunity due to T reg cell deficiency. These results suggest that Satb1-dependent T reg-SE activation is crucial for T reg cell lineage specification in the thymus and that its perturbation is causative of autoimmune and other immunological diseases.