In lung transplantation (LT), alloreactive T cell priming begins in the graft, a process that can be inhibited by seeding the graft with recipient-derived expanded polyclonal regulatory T cells (Tregs) using ex vivo lung perfusion (EVLP) prior to transplantation. However, this therapy alone cannot attenuate acute rejection in non-immunosuppressed animals. Interleukin 2 (IL-2)-anti-IL-2 antibody complexes (IL-2 C) promote Treg expansion in vivo with concomitant tacrolimus (Tac) administration. We combined IL-2 C and Tac with pre-transplant Treg administration during EVLP in a Fischer 344 to Wistar Kyoto rat LT model, to test the hypothesis that this strategy would facilitate intragraft Treg expansion and function. Recipients were given no treatment, Tac alone, IL-2 C/Tac alone, Treg alone, or Treg/IL-2 C/Tac. After 7 days, graft CD25Foxp3 content increased as a result of Treg therapy, and cellular rejection was attenuated in the IL-2 C/Tac and Treg/IL-2 C/Tac groups. Graft Treg content and Treg-to-effector T cell ratio (Treg/Teff) at day 7 was highest in animals receiving Treg/IL-2 C/Tac, which has important implications for long-term immunomodulation. Our data suggest that pre-transplant administration of graft-directed Treg cell therapy combined with Treg-permissive immunosuppression may be a viable therapeutic approach to modulate rejection in LT.