Natural killer (NK) cells can kill a wide range of cancer cells and are a promising tool for cell therapy of cancer. NK cells cytotoxicity is regulated by a balance between stimulatory and inhibitory signals. Interleukin-2 is known to increase NK cell cytotoxicity. Although many cytokines have been studied in efforts to induce durable NK cell expansions, most reports indicate a rather modest effect and the requirement for additional stimuli. We found that contact with the K562 myeloid leukemia cell line, genetically modified to express a membrane-bound form of interleukin-15 and the ligand for the costimulatory molecule 4-1BB, induced vigorous expansion of NK cells from peripheral blood. Based on these findings, we developed a method for large-scale clinical-grade expansion of NK cells. This method is currently used to expand allogeneic NK cells for infusion in patients with leukemia and solid tumors. We here summarize methods for expansion and activation of NK cells from human peripheral blood mononuclear cells as well as clinical-scale methods to produce NK cells for immunotherapy under Current Good Manufacturing Practices (cGMP) conditions.