Aim Everolimus (EVL) is an mTOR-inhibitor and a modified form of Sirolimus (SRL) which is used clinically in combination with Mycophenolate and/or Tacrolimus (TAC). The aim was to tests the effects of EVL alone or in combination with Mycophenolic acid (MPA), the in vivo active form of Mycophenolate or TAC on the generation of regulatory T cells (Tregs) using the Treg-MLR previously described. Methods Therapeutic or sub-therapeutic drug concentrations of EVL, MPA and TAC and alone or in combination were added to MLRs of healthy volunteers, assessing proliferation by 3H-TdR incorporation and newly generated CD4 + CD127 − CD25 High FOXP3 + (nTregs) and CD4 + CD127 − CD25 + FOXP3 + (tTregs) in CFSE labeled responder cells by flow cytometry. Results In comparison to medium controls, EVL dose-dependently inhibited proliferation but amplified the generation of Treg subsets in HLA 2-DR matched and mismatched MLRs ( n = 10) with similar effects seen testing MPA. However, TAC inhibited both the proliferation and generation of Tregs confirming our previous results. Combinations of EVL + MPA inhibited proliferation and amplified the generation of both nTregs and tTregs in an additive manner, when compared to medium controls or each drug tested alone ( p Conclusions In conclusion, EVL has the same regulatory mechanism of action as SRL we tested previously. Since EVL does not have the black box warning unlike SRL against its use in liver transplant patients (LT), EVL may be substituted for its tolerogenic effects in LT studies.