Neutrophilic in vitro differentiation showed recapitulation of Kostmann phenotype and rescue upon genetic correction. (A) Schematic overview of the hematopoietic in vitro differentiation protocol. Spin-EBs were generated, selected, and transferred into plates for adhesion. Attached EBs formed myeloid cell–forming complexes that released progenitors as suspension cells into the medium. After hematopoietic specification, step suspension cells were collected and applied to neutrophilic differentiation. IL-3, interleukin-3. (B-C) After neutrophilic differentiation, morphology was assessed by (B) May-Grünwald Giemsa staining of cytospins. Original magnification ×200 (insets, ×400). (C) Percentages of neutrophilic granulocytes (Gran), promyelocytes (Prom) and monocytes (Mono) are presented as mean ± standard deviation (SD) of n = 10 independent experiments. (D) Percentage (mean ± SD) of CD15⁺ (neutrophilic marker) HAX1^WT/WT, HAX1^W44X, and HAX1^corrected cells after neutrophilic differentiation as assessed by flow cytometry (n = 4). (E) Representative fluorescence-activated cell sorted plots and gating strategy are shown. The values indicate mean ± SD of n = 4 experiments. (F) DNA staining after PMA stimulation showed NET formation in peripheral blood granulocytes, HAX1^WT/WT and HAX1^corrected-iPSC–derived cells. Original magnification ×100 (insets, ×400); SYTOX Green stain. (C-D) **P < .01 when compared with HAX1^W44X-iPSCs.