Adoptive natural regulatory T cell (nTreg) therapy has improved the outcome for patients suffering from graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (Allo-HCT). However, fear of broad immune suppression and subsequent dampening of beneficial graft-versus-leukemia (GVL) responses remains a challenge. To address this concern, we generated alloreactive induced Tregs (iTregs) from resting CD4⁺ or CD8⁺ T cells and tested their ability to suppress GVH and maintain GVL responses. We utilized major mismatched and haploidentical murine models of HCT with host-derived lymphoma or leukemia cell lines to evaluate GVH and GVL responses simultaneously. Alloreactive CD4⁺ iTregs were effective in preventing GVHD, but abrogated the GVL effect against aggressive leukemia. Alloreactive CD8⁺ iTregs moderately attenuated GVHD while sparing the GVL effect. Hence, we reasoned that using a combination of CD4⁺ and CD8⁺ iTregs could achieve the optimal goal of Allo-HCT. Indeed, the combinational therapy was superior to CD4⁺ or CD8⁺ iTreg singular therapy in GVHD control; importantly, the combinational therapy maintained GVL responses. Cellular analysis uncovered potent suppression of both CD4⁺ and CD8⁺ effector T cells by the combinational therapy that resulted in effective prevention of GVHD, which could not be achieved by either singular therapy. Gene expression profiles revealed alloreactive CD8⁺ iTregs possess elevated expression of multiple cytolytic molecules compared to CD4⁺ iTregs, which likely contributes to GVL preservation. Our study uncovers unique differences between alloreactive CD4⁺ and CD8⁺ iTregs that can be harnessed to create an optimal iTreg therapy for GVHD prevention with maintained GVL responses.