Cord blood (CB)-derived chimeric antigen receptor (CAR) natural killer (NK) cells have demonstrated significant antitumor efficacy. We recently reported that CB-derived CAR NK cells predominantly originate from CD7CD56CD34HLA-DRLin NK cell precursors in CB. Here, we demonstrate that stimulating the interleukin (IL)-15 receptor on these NK precursors enhances the production of CAR NK cells from CB cells. In CB CD56CD34HLA-DRLin cells, the IL-15 receptor was exclusively expressed on CD7 NK cell precursors. Using K562 feeder cells that express not only 4-1BB ligand and membrane-bound (mb) IL-21 but also mbIL-15 significantly increased the production of mature NK cells from the purified NK cell precursors or T cell-depleted CB cells. The in vitro and in vivo antitumor effects of CAR NK cells generated using K562 feeder cells that express mbIL-15 were comparable to those of CAR NK cells produced using K562 feeder cells that do not express mbIL-15. These results suggest that K562 feeder cells expressing 4-1BBL, mbIL-21, and mbIL-15 can increase the production of CAR NK cells from CB cells while maintaining their cytotoxic potential. This method could also be useful for expanding NK cells from CB for any type of adoptive NK cell therapy with or without CAR transduction.