GMP deficient for Btk exhibit a decreased differentiation potential and drive myeloid development toward the granulocytic lineage. A total of 500 GMP sorted out of individual wild-type (wt) and Btk-ko mice were seeded in methylcellulose media supplemented with SCF, IL-3, and GM-CSF. The experiment was performed in quadruplicate. (A) After 6 days in culture, colony-forming units (CFU) were counted. Data presented are the mean values (± SD). (B) The cell number per CFU was calculated. (C) One CFU with a representative size generated from wild-type (wt) or Btk-deficient GMP was photographed and is presented. Scale bars represent 200 μm. (D) Differentiated cells were analyzed by flow cytometry at day 8 of culture. Data presented are the mean (± SD) of cell numbers positive either for the surface marker CD11b⁺/Gr-1⁻/F4/80⁻ (undifferentiated cells), for CD11b⁺/Gr-1⁺ (granulocytes), or for CD11b⁺/F4/80⁺ (monocytes). (E) Twenty to thirty individual CFU per biological replicate were processed for cytospins and Pappenheim staining and analyzed for the cell content by morphology. CFU-M corresponds to more than 90% of the cells were macrophages, CFU-G corresponds to more than 90% of cells were neutrophils, CFU-GM corresponds to the content of macrophages and granulocytes was in between 20 to 80%. (F) A representative cytospin per analyzed genotype is presented. Scale bar represents 50 μm. In panel E the data are presented as mean values (± SEM). Data were analyzed with Student t test. *P ≤ 0.05, ***P ≤ 0.0005. n represents the number of biological replicates.