Natural killer (NK) cell-based adoptive immunotherapy is an attractive adjuvant treatment option for patients with acute myeloid leukemia. Recently, we reported a clinical-grade, cytokine-based culture method for the generation of NK cells from umbilical cord blood (UCB) CD34⁺ hematopoietic progenitor cells with high yield, purity and in vitro functionality. The present study was designed to evaluate the in vivo anti-leukemic potential of UCB-NK cells generated with our GMP-compliant culture system in terms of biodistribution, survival and cytolytic activity following adoptive transfer in immunodeficient NOD/SCID/IL2Rg(null) mice. Using single photon emission computed tomography, we first demonstrated active migration of UCB-NK cells to bone marrow, spleen and liver within 24 h after infusion. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Thereafter, we showed that low dose IL-15 mediates efficient survival, expansion and maturation of UCB-NK cells in vivo. Most importantly, we demonstrate that a single UCB-NK cell infusion combined with supportive IL-15 administration efficiently inhibited growth of human leukemia cells implanted in the femur of mice, resulting in significant prolongation of mice survival. These preclinical studies strongly support the therapeutic potential of ex vivo-generated UCB-NK cells in the treatment of myeloid leukemia after immunosuppressive chemotherapy.