In addition to Foxp3+ CD4+ regulatory T cells (CD4+ T reg cells), Foxp3− CD8+ regulatory T cells (CD8+ T reg cells) are critical to maintain immune tolerance. However, the molecular programs that specifically control CD8+ but not CD4+ T reg cells are largely unknown. Here, we demonstrate that simultaneous disruption of both TGF-β receptor and transcription factor Eomesodermin (Eomes) in T cells results in lethal autoimmunity due to a specific defect in CD8+ but not CD4+ T reg cells. Further, TGF-β signal maintains the regulatory identity, while Eomes controls the follicular location of CD8+ T reg cells. Both TGF-β signal and Eomes coordinate to promote the homeostasis of CD8+ T reg cells. Together, we have identified a unique molecular program designed for CD8+ T reg cells.