Increased NK lineage restriction during progression through sequential NK cell stages along the in vitro developmental trajectory. (A) Schematic diagram summarizes experimental outline: progeny from in vitro cultured Lin⁻CD34⁺ progenitors after 2 weeks were sorted into S1/2 (Lin⁻CD34⁺CD161⁻CD56⁻CD94⁻), S3a (Lin⁻CD34⁻CD161⁺CD45RA⁺CD56⁻CD94⁻), S3b (Lin⁻CD34⁻CD161⁺CD56⁺NKp44⁺⁻CD94⁻), and S4 (Lin⁻CD34⁻CD161⁺CD56⁺CD94⁺) populations, replated, and lineage output analyzed after 2 and 3 weeks. (B) Representative FACS profiles showing gating scheme of different lineage output after 3-week subculture: Monocyte/DC (CD19⁻CD14⁺CD11c⁺), B (CD14⁻CD11c⁻CD19⁺), CD56⁺ ILCs (CD14⁻CD11c⁻CD19⁻CD56⁺), which were further separated into RORγt⁻ NK and RORγt⁺ ILC3 cells. (C) Lineage output generated from CD34⁺, S1/2, S3a, S3b, and S4 populations after 2 and 3 weeks in subculture (3 individual donors, 3 replicates for each donor). (D) Summary of lineage-output scores aggregated across all the donors and time points, compared between sorted populations as in panel C. (E) Lineage output within CD56⁺ cells generated after 2 and 3 weeks from CD34⁺, S1/2, S3a, S3b, and S4 populations categorized as either wells contained both RORγt⁻ and RORγt⁺ (NK_ILC3), or only RORγt⁻ cells (NK_only; 3 individual donors, 3 replicates for each donor). (F) Summary of CD56⁺ ILC lineage-output scores aggregated across all the donors and time points, compared between sorted populations as in panel E. The lineage scoring criteria are described in detail in supplemental Methods. For panels C-F, statistical significance was performed with Kruskal-Wallis test between pairwise comparisons among S3a subset vs the other 4 subsets; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001; and n.s., not significant.