Graft-versus-host disease (GVHD), leukemia relapse, and immune deficiency remain the major limitations of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor natural killer cells (NK) and cytokines have good potential in GVHD prevention and immune reconstitution enhancement. Improved survival after allo-HSCT therefore requires effective prophylaxis to reduce GVHD and strategy to mediate graft-versus-leukemia (GVL) effect. We studied the administration of expanded donor NK cell infusion and interleukin-2 (IL-2) and IL-15 mixture treatment in a murine allo-HSCT model for its effects on GVHD, immune reconstitution and leukemia relapse. In the GVHD model, recipient mice were reconstituted with bone marrow (BM) cells and splenocytes via vein. In the leukemia model, recipient mice were inoculated with EL9611 leukemia cells via vein 8 d prior to transplant. NK cell infusion mice group had lower clinical GVHD scores and suffered less severe GVHD-associated weight loss than control mice group. 90% of control mice died of leukemia relapse within 52 d post-transplant. NK cell infusion and IL-2 and IL-15 treatment recipient mice had improved survival compared with control mice. NK cell infusion and IL-2 and IL-15 treatment recipient mice had also accelerated lymphoid immune reconstitution compared with control mice group. Expanded donor NK cell infusion and IL-2 and IL-15 treatment could promote lymphoid immune reconstitution, mitigate GVHD, and reduce leukemia relapse in allo-HSCT recipients. The findings may have important significance for complication prevention in clinical allo-HSCT.