Study concept. In clinical practice, early-gestation IUHCT affords the lowest fetal immune barrier but is impeded by higher technical difficulty and fewer treatable patients. Late-gestation IUHCT, in contrast, affords lower technical difficulty and more treatable patients, but is impeded by a higher immune barrier leading to allograft rejection. To study this problem, we employed murine models of early-gestation IUHCT (injection of allogeneic hematopoietic cells before birth at 14 DPC) and late-gestation IUHCT (injection of allogeneic hematopoietic cells after birth at 20 DPC). First, we investigated which regulatory T-cell populations, if any, are elevated after early-gestation IUHCT and may contribute to alloengraftment and tolerance. Next, we validated our model of late-gestation IUHCT, showing it to reliably lead to allograft rejection. Next, we tested whether IUHCT-induced Tregs or naive donor Tregs (a clinically translatable approach) could prevent allograft rejection in our model of late-gestation IUHCT. Outcomes assessed included long-term, multilineage chimerism, donor-specific tolerance, and the absence of graft-versus-host disease.