Selective depletion (SD) of host-reactive donor T cells from allogeneic stem-cell transplants (SCTs) using an anti-CD25 immunotoxin (IT) is a strategy to prevent acute graft-versus-host disease (aGvHD). There is concern that concurrent removal of regulatory T cells (T_regs) with incomplete removal of alloactivated CD25⁺ T cells could increase the risk of aGvHD. We therefore measured T_regs in the blood of 16 patients receiving a T-cell–depleted allograft together with anti–CD25-IT–treated SD lymphocytes, in 13 of their HLA-identical donors, and in 10 SD products. T_regs were characterized by intracellular staining for forkhead box protein 3 (FOXP3) and by quantitative reverse-transcription–polymerase chain reaction (qRT-PCR) for FOXP3 gene in CD4⁺ cells. Patients received a median of 1.0 × 10⁸/kg SD T cells and a stem cell product containing a median of 0.25 × 10⁴/kg residual T cells. T_regs reconstituted promptly after SCT and underwent further expansion. Of the CD4⁺ T cells in SD products, 1.5% to 4.8% were CD25⁻ T_regs. Acute GvHD (≥ grade II) was restricted to 5 patients whose donors had significantly (P = .019) fewer T_regs compared with those without clinically significant aGvHD. These results suggest that rapid T_reg reconstitution can occur following SD allografts, either from CD25⁻ T_regs escaping depletion, or from residual CD25⁻ and CD25⁺ T_regs contained in the stem-cell product that expand after transplantation and may confer additional protection against GvHD.