Culture of CD34⁺/Lin⁻/DR⁻ cells in stromal-based long-term NK cell culture with IL-2 alone results in NK cell differentiation without maintenance of CD34⁺ cells, committed myeloid progenitors, or lymphoid progenitors capable of reinitiating long-term NK cell cultures. A sequential culture was designed to induce commitment and further differentiation of NK cell progenitors from cultures, which maintain LTC-IC. Fresh CD34⁺/Lin⁻/DR⁻ cells are plated in Transwell inserts above allogeneic, irradiated stroma in LTC medium supplemented with IL-3 and MIP-1α. After 14 days, progeny are harvested and plated in direct contact with stroma in the presence or absence of indicated cytokines for “NK cell progenitor switch” conditions. After 2 weeks, medium was removed without loss of nonadherent cells then replated in NK medium with IL-2 alone to further differentiate NK cells.