Mouse renal allografts have a remarkable ability to promote acceptance across full major histocompatibility complex incompatibilities in certain strain combinations without immunosuppression. The mechanism is unknown but is believed to involve immunoregulation. This study tests whether Foxp3⁺ T-regulatory cells are responsible in the early phase of graft acceptance, using B6.Foxp3^DTR mice that express diphtheria toxin receptor (DTR) in Foxp3⁺ cells. The administration of DT to B6.Foxp3^DTR recipients with accepted DBA/2 kidneys, 3 weeks to 3 months after transplantation, caused a marked depletion of Foxp3 cells and triggered acute cellular rejection, manifested by a sudden increase in blood urea nitrogen within a week. None of the controls showed an increase in blood urea nitrogen, including DT-treated B6 wild-type recipients of DBA/2 kidneys or B6.Foxp3^DTR recipients of isografts. Accepted DBA/2 allografts showed prominent lymphoid sheaths around arteries containing numerous CD3⁺Foxp3⁺ cells, CD4⁺ cells, dedritic cells, and B cells, which was independent of CCR4. The lymphoid sheaths disintegrate after Foxp3 depletion, accompanied by widespread CD8 interstitial mononuclear inflammation, tubulitis, and endarteritis. The Foxp3 depletion caused an increased frequency of donor-reactive cells in the spleen by interferon (IFN) γ enzyme-linked immunosorbent spot (ELISPOT) assays and increased expression of the maturation markers, CD86 and IA^b, on dendritic cells in the spleen and kidney. We conclude that Foxp3⁺ cells are needed to maintain acceptance of major histocompatibility complex–incompatible renal allografts in the first 3 months after transplantation and may act by inhibiting DC maturation.