CD4+ T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4+ CD25+ FOXP3+ Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg. This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4+, especially CD4+ CD25+ T cells. CD4+ T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100 days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4+ T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4+ CD25+ T cells' response to rIL-5 and expression of IL-5Rα was also assessed. rIL-5 was sufficient to promote transplant tolerance mediating CD4+ T cells' survival in culture with specific-donor alloantigen. Tolerant CD4+ T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4+ T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4+ CD25+ T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4+ CD25+ T cells expressed IL-5Rα. This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4+ CD25+ T cells that mediate transplant tolerance.