Fetal liver Lin⁻CD3⁻CD122⁺NK1.1⁻DX5⁻ NK-cell progenitors efficiently generate NK and T cells in vitro. Fifteen to thirty and 5 Lin⁻CD3⁻CD122⁺NK1.1⁻DX5⁻ NKPs as well as 100 Lin⁻ cells (control) sorted from the livers of 14.5- and 18.5-day-old fetuses were cultured on OP9 and OP9-DL1 stroma cell lines with the following cytokines: IL-7 (first week only), KL, FL, IL-2, and IL-15. After 21 days, cells were harvested and evaluated for the presence of NK (TCRβ-NK1.1⁺DX5⁺ or TCRβ⁻NKp46⁺), B (CD19⁺), T (NK1.1⁻CD25⁺Thy1.2⁺, or NK1.1⁻CD25⁺Thy1.2⁺TCRβ⁺) and NK1.1⁺ T cells (TCRβ⁺NK1.1⁺) by FACS. TO-PRO-1 was used to exclude dead cells, and GFP was used to exclude stroma cells from the analysis. (A) Representative FACS profiles of cells generated from fetal liver E14.5 NKPs and Lin⁻ cells cultured on OP9 and OP9-DL1 stroma. The specific gates are indicated in the text above the plot and by arrows. (B) Mean ± SD proportion of total positive wells and wells containing NK cells generated from 15 to 30 fetal liver E14.5 NKPs cultured on OP9 (white bars) and OP9-DL1 (black bars) stroma. Data represent mean ± SD values from 3 independent experiments (2 with OP9-DL1 and 1 with OP9 stroma) with 17-24 wells analyzed in each experiment (total 58 wells). (C) Representative FACS profiles of cells generated from fetal liver E18.5 NKPs and Lin⁻ cells cultured on OP9 and OP9-DL1 stroma. The specific gates are indicated in the text above the plot and by arrows. Mean ± SD proportion of total positive wells and wells containing NK, B, T, and NK1.1⁺ T cells generated from 5 fetal liver NKPs cultured on (D) OP9 and (E) OP9-DL1 stroma. Data represent mean ± SD values from 5 independent experiments with 24-72 wells analyzed in each experiment (total 220 wells).