Regulatory T cells (Tregs) play a significant role in immune tolerance. Since Treg function deeply depends on Interleukin‐2 signaling, calcineurin inhibitors could affect their suppressive potentials, whereas mammalian target of rapamycin (mTOR) inhibitors may have less impact, as mTOR signaling is not fundamental to Treg proliferation. We previously reported a novel mixed hematopoietic chimerism induction regimen that promotes Treg proliferation by stimulating invariant natural killer T cells under CD40 blockade. Here, we use a mouse model to show the impact of tacrolimus (TAC) or everolimus (EVL) on the establishment of chimerism and Treg proliferation in the regimen. In the immunosuppressive drug‐dosing phase, peripheral blood chimerism was comparably enhanced by both TAC and EVL. After dosing was discontinued, TAC‐treated mice showed gradual graft rejection, whereas EVL‐treated mice sustained long‐term robust chimerism. Tregs of TAC‐treated mice showed lower expression of both Ki67 and cytotoxic T lymphocyte antigen‐4 (CTLA‐4), and lower suppressive activity in vitro than those of EVL‐treated mice, indicating that TAC negatively impacted the regimen by interfering with Treg proliferation and activation. Our results suggest that the usage of calcineurin inhibitors should be avoided if utilizing the regimen to induce Tregs in vivo for the establishment of mixed hematopoietic chimerism.