Defined cytokines and direct contact with stroma are sufficient for survival of NK progenitors. Ten thousand CD34⁺/Lin⁻/DR⁻ cells were plated in direct contact with stroma (black bars) or suspended above stroma in a Transwell insert (white bars) in hydrocortisone containing LTC medium and the indicated cytokines (n = 4). After 5 weeks, conditions were switched to those favoring NK cell terminal differentiation. Starting CD34⁺/Lin⁻/DR⁻ cells plated in direct contact with stroma were switched by removal of all LTC medium, nonadherent cells were pelleted, resuspended in NK medium, and replated on the initial stroma layer. Progeny of CD34⁺/Lin⁻/DR⁻ cells cultured in a Transwell were harvested, resuspended in NK medium, and replated on the stromal layer below. After 4 to 5 weeks in NK medium, cells were harvested, counted, and phenotyped. NK cells resulting from this final step defined NK cell progenitors surviving conditions during the first culture period. IL-2 + IL-7 in direct contact with stroma ligands resulted in significantly greater NK cell progenitor maintenance compared with no cytokines (P = .025) or the same cytokines without contact with stroma ligands (P = .019).