Tolerogenic dendritic cells (Tol-DCs) and regulatory T cells (Treg) are key factors in the induction and maintenance of transplantation tolerance. We previously demonstrated that ex vivo-isolated Tol-DCs promote Treg generation, and vice versa, in an in vitro co-culture system. Here we demonstrate the occurrence of such an immune regulatory feedback loop in vivo. Tol-DC generated in vitro by treatment with LF 15-0195 exhibited features of immature DC and express low levels of MHC class II, CD86 and CD40. These Tol-DCs were capable of augmenting CD4(+)CD25(+)CTLA4(+) and FoxP3(+) Treg cell numbers and activity in cardiac allograft recipients. On the other hand, Tol-DCs possessed an ability to generate Treg cells in vitro. The adoptive transfer of these in vitro-generated Treg cells resulted in an increase of Tol-DC in vivo, suggesting that an immune regulatory feedback loop, between Tol-DC and Treg, exists in vivo. Furthermore, the administration of in vitro-generated Tol-DCs or Treg cells prevented rejection of allografts. Co-administration of Tol-DC and Treg synergized efficacy of promoting allograft survival heart transplantation. The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs and Treg.