Protocols to use Foxp3+ T-regulatory (Treg) cells for cellular therapy, especially postallogeneic stem cell transplantation, are currently being developed and tested by various groups. Inhibitors of DNA methyltransferase (Dnmt) enzymes have been advocated as a means to promote and stabilize Foxp3 expression in Tregs undergoing expansion in vitro before their injection in vivo. We investigated the effects of conditionally deleting two Dnmt enzymes that co-immunoprecipitated with Foxp3 in Treg isolates. Deletion of Dnmt1, but not Dnmt3a, decreased the numbers and function of peripheral Tregs and impaired conversion of conventional T cells into Foxp3+ Tregs under polarizing conditions. Importantly, mice with conditional deletion of Dnmt1 in their Tregs died of autoimmunity by 3 to 4 weeks of age unless they were rescued by perinatal transfer of wild-type Tregs. Conditional Dnmt1 deletion did not affect methylation of CpG sites within Foxp3 but decreased global DNA methylation and altered Treg expression of several hundred pro-inflammatory and other genes. Hence, Dnmt1 is necessary for maintenance of the core gene program underlying Treg development and function, and its deletion within the Treg lineage leads to lethal autoimmunity. These data suggest that caution may be warranted when considering the use of DNMT inhibitors in development of Treg-based cellular therapies.