Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4+CD25 + regulatory T cells (nTregs) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nTregs has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nTregs could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nTregs in UCB units is limited, and average yield after expansion was only 1 × 109 nTregs. Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nTregs. PB nTregs were purified under GMP conditions and expanded 80-fold to yield 19 × 10 9 cells using anti-CD3 antibody-loaded, cell-based artificial antigen-presenting cells (aAPCs) that expressed the high-affinity Fc receptor and CD86. A single restimulation increased expansion to ∼3000-fold and yield to >600 × 109 cells while maintaining Foxp3 expression and suppressor function. nTreg expansion was ∼50 million-fold when flow sort-purified nTregs were restimulated four times with aAPCs. Indeed, cryopreserved donor nTregs restimulated four times significantly reduced GVHD lethality induced by the infusion of human T cells into immune-deficient mice. The capability to efficiently produce donor cell banks of functional nTregs could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.