Previously, we showed that human umbilical cord blood (UCB) regulatory T cells (Tregs) could be expanded approximately 100-fold using anti-CD3/28 monoclonal antibody (mAb)-coated beads to provide T-cell receptor and costimulatory signals. Because Treg numbers from a single UCB unit are limited, we explored the use of cell-based artificial antigen-presenting cells (aAPCs) preloaded with anti-CD3/28 mAbs to achieve higher levels of Treg expansion. Compared with beads, aAPCs had similar expansion properties while significantly increasing transforming growth factor β (TGF-β) secretion and the potency of Treg suppressor function. aAPCs modified to coexpress OX40L or 4-1BBL expanded UCB Tregs to a significantly greater extent than bead- or nonmodified aAPC cultures, reaching mean expansion levels exceeding 1250-fold. Despite the high expansion and in contrast to studies using other Treg sources, neither OX40 nor 4-1BB signaling of UCB Tregs reduced in vitro suppression. UCB Tregs expanded with 4-1 BBL expressing aAPCs had decreased levels of proapoptotic bim. UCB Tregs expanded with nonmodified or modified aAPCs versus beads resulted in higher survival associated with increased Treg persistence in a xenogeneic graft-versus-host disease lethality model. These data offer a novel approach for UCB Treg expansion using aAPCs, including those coexpressing OX40L or 4-1 BBL.