The management of acute lymphoblastic leukemia (ALL) patients has witnessed profound changes in recent years. Nonetheless, most patients tend to relapse, underlining the need for new therapeutic approaches. The anti-leukemic potential of natural killer (NK) cells has over the years raised considerable interest. In this study, we developed an efficient method for the expansion and activation of NK cells isolated from healthy donors and ALL patients for clinical use. NK cell products were derived from peripheral blood mononuclear cells of 35 healthy donors and 4 B-lineage ALL by immunomagnetic CD3 T cell depletion followed by CD56 cell enrichment. Isolated NK cells were expanded and stimulated in serum-free medium supplemented with irradiated autologous feeder cells and autologous plasma in the presence of clinical grade interleukin (IL)-2 and IL-15 for 14 days. Healthy donor NK cells expanded on average 34.9 ± 10.4 fold and were represented, after expansion, by a highly pure population of CD3(-)CD56(+) cells showing a significant upregulation of natural cytotoxicity receptors, activating receptors and maturation markers. These expanded effectors showed cytolytic activity against K562 cells and, most importantly, against primary adult B-lineage ALL blasts. NK cells could be efficiently isolated and expanded-on average 39.5 ± 20.3 fold-also from primary B-lineage ALL samples of patients in complete remission. The expanded NK cells from these patients showed a significantly increased expression of the NKG2D- and DNAM1-activating receptors and were cytotoxic against K562 cells. These data provide the basis for developing new immunotherapeutic strategies for the management of ALL patients.