BACKGROUND: Allogeneic natural killer (NK) cell adoptive immunotherapy is a growing therapeutic option for patients. Clinical-scale production of NK cells using immunomagnetic selection complies with current good manufacturing practices (cGMPs) and allows for closed-system, automated purification. We report our experience with CD3/CD19 cell-depleted (CD3/CD19dep) NK cell production and compare to previous methods of CD3 cell depletion and CD3 cell depletion/CD56 cell enrichment. STUDY DESIGN AND METHODS: Nonmobilized mononuclear cells collected by apheresis were incubated with anti-CD3/anti-CD19 microbeads and depleted in an automated cell selection system (CliniMACS, Miltenyi). The NK cell–enriched products were incubated overnight in interleukin (IL)-2 or IL-15, washed, and resuspended prior to lot release testing and infusion. RESULTS: Since 2010, 94 freshly infusible CD3/CD19dep NK cell products were manufactured in support of eight clinical trials. Sixty-six products were incubated in IL-2 and 28 products in IL-15. Processing resulted in a mean NK cell recovery of 74% and viability of 95.8%; NK cells, T cells, B cells, and monocytes accounted for 47%, 0.2%, 0.08%, and 49% of the final products, respectively. Seven products required dose adjustments to meet lot release. The specification for purity changed throughout the evolution of manufacturing. IL-2 or IL-15 activation enhanced in vitro cytotoxicity compared to preactivated cells. There was no difference in final product composition or cytotoxicity between cytokine cohorts. CONCLUSION: Clinical-scale/cGMP production of NK cells using CD3/CD19 cell-depletion effectively minimized T-cell and B-cell contamination in a single manipulation without compromise to NK-cell recovery. Cytokine activation increased in vitro cytotoxicity compared to column-depleted, preactivated NK cells.