In vitro T-cell differentiation of CD34⁺CD3^– hematopoietic stem and progenitor cells (HSPCs). (A) Representative analysis of T-cell differentiation of control and P2 HSPCs after 6 weeks of culture in an ATO system. The fluorescence-activated cell sorter (FACS) plots show expression of early and late markers of T-cell differentiation upon gating on LIVE/DEAD^–CD45⁺CD14^–CD56^–CD34^– cells. (B) The bar graph shows the absolute cell counts per ATO of indicated cell subsets in control (gray bar) and P2 (red bar) samples analyzed in parallel. (C) The histograms show distribution of cells in the different phases of cell cycle after cell staining for DNA content (DAPI) in control and P2, upon gating on total CD45⁺CD56^– cells. (D) The bar graph shows the frequency of apoptotic cells after staining with annexin V and DAPI in control and P2 upon gating on total CD45⁺CD56^– cells. (E-F) FACS plot representing CXCR4 and CD98 expression in more immature (CD5⁺CD7^hi or CD1a⁺CD7⁺) and more mature (CD5⁺CD7^lo or CD1a⁺CD7^–) developing T-cell progenitors and in mature TCRαβ⁺CD3⁺ cells.