TOX2 expression increases during NK cell development. (A) Peripheral blood mononuclear cells collected from healthy donors were subjected to flow-based cell sorting for NK cells (CD3⁻CD56⁺), NKT cells (CD3⁺CD56⁺), T cells (CD3⁺CD56⁻), B cells (CD3⁻CD19⁺), and monocytes (CD14⁺). The expression of TOX2 was analyzed by RT-qPCR (n = 3) *P < .005, **P < .001. (B) Different developmental stages of NK cells were isolated by flow sorting from normal BM samples. The expression of TOX2 and a panel of genes involved in NK cell development were analyzed by RT-qPCR (n = 3). (C) Different developmental stages appeared during in vitro NK cell development from UCB-derived CD34⁺ cells and were analyzed by flow cytometry at different time points (day 2, day 7, and day 17). Results are representative of 4 independent experiments. (D) Different developmental cell populations were flow sorted at various time points: a mixed population consisting of HSCs, stage I and stage II on day 2, stage III on day 7, and stage IV and stage V on day 17. RNA samples were collected from the sorted cells for cDNA synthesis. RT-qPCR was performed to analyze the expression of TOX2 and a panel of NK cell developmental genes (n = 4).