Notch-independent generation of thymic and BM NK cells from Lin⁻CD122⁺NK1.1⁻DX5⁻ NK/T progenitors. Single Lin⁻CD122⁺NK1.1⁻DX5⁻ NKPs from BM from 10- to 12-week-old mice and single DN1 (Lin⁻CD25⁻Kit^high) cells from the thymus from 6- to 8-week-old mice were directly sorted onto OP9DL1 and OP9 stroma layers and cultured for 21 days with KL, FL, and IL-15. Clones were harvested and analyzed by FACS for the presence of BM-dependent NK (TCR-β⁻CD25⁻NK1.1⁺CD122⁺IL-7Rα⁻) and thymic NK (tNK) cells (TCR-β⁻CD25⁻Ly49D⁻NK1.1⁺CD122⁺IL-7Rα⁺), as well as TCR-β⁺CD25⁻NK1.1⁻CD122⁻ T cells. (A) Representative FACS profiles of clones generated from single NKPs and DN1 thymocytes cultured on OP9DL1 stroma. Text above panels indicates prior gating. (B) Mean (SD) proportion of total generated clones and clones containing BM and thymic NK cells and T cells from single NKPs cultured on OP9DL1 and OP9. (C) Mean (SD) proportion of total generated clones and clones containing BM and thymic NK cells and T cells from single DN1 cells cultured on OP9DL1 and OP9. Data expressed in relation to plated single cells, from 2 to 4 independent experiments, were 24 to 40 clones analyzed in each experiment. *P < .05; **P < .01; ***P < .001.