Pre-NKP and rNKP are lineage restricted and give rise only to NK cells in vitro. (A) Pre-NKP and rNKP lack B-cell potential in vitro. Individual wells with OP9 cells were seeded with 10 indicated progenitor cells and cultured in the presence of SCF, IL-7, Flt3 ligand, and IL-15. Wells were analyzed by flow cytometry 10 days later. No wells were positive for Gr-1⁺CD11b⁺ myeloid cells (data not shown). (B) Pre-NKP and rNKP lack in vitro T-cell potential. Flow cytometry analysis of the lineage output of individual wells seeded on OP9-DL1 cells with 10 indicated progenitor cells. No wells analyzed were positive for myeloid cells (data not shown). Data are representative of 1 of 4 experiments. (C) Pre-NKP and rNKP generate NK cells at a high clonal frequency. Number of wells plated with single cell and the number of wells positive for NKp46⁺ cells after 10 days. Data are representative of 1 of 3 experiments.