Background: It has been shown that ex vivo-expanded regulatory T cells (Treg) can promote transplant tolerance in small animal models as well as in phase I clinical trials of hematopoietic stem cell transplantation. The issue has been raised of establishing Treg banks for adoptive cell therapy of organ transplant rejection and autoimmune disease. However, little is known about the persistence, stability and fate of infused Treg, especially third-party allogeneic Treg, and the influence of T cell-depleting and other immunosuppressive agents on their survival and therapeutic efficacy. Methods and Results: In this study, we flow-sorted CD4+CD25+CD127- Treg from normal juvenile cynomolgus monkey PBMC and expanded these cells using a protocol with artificial antigen-presenting cells. Three rounds of stimulation expanded the cells over 1000-fold and consistently generated over 108 Treg from 30 ml peripheral blood, while maintaining expression of Foxp3, CD25, CTLA-4, Helios; potent capacity to suppress T cell proliferation in vitro; demethylation status at the Treg-Specific Demethylation Region (TSDR), and down-regulated expression of CD45RA, CCR7, CD62L and upregulation of CXCR3 suggesting that these cells are likely to migrate to inflamed sites immediately after infusion in vivo. More importantly, when CFSE-labeled autologous (auto) and VPD450-labeled allogeneic (allo) ex vivo expanded Tregs were transferred simultaneously into monkeys, the number of auto-Treg was up to 100 per ml peripheral blood for at least 6 days (ie. for the duration of the experiment) in both control and ATG/rapamycin-treated (IS) monkeys. In contrast, by day 3 after their infusion, allo-Treg could not be detected in control monkeys, while they were maintained up to 100 cells per ml peripheral blood for at least 15 days in IS monkeys. These Tregs maintain high expression of Foxp3, CD25, and CXCR3 expression, and low expression of CD127, CCR7, and CD62L. Conclusion: The finding that allo-Treg can survive in IS-treated monkeys and maintain Treg phenotype supports the concept and potential utility of Treg banks for clinical application in transplantation and autoimmune disease and provides valuable information required to take allo-Treg therapy towards phase II/III clinical trials for efficacy assessment.