PIK3CD GOF mutations block B cell development in the bone marrow at the pre-BII stage. BM aspirates from healthy donors (n = 6), patients with PIK3CD GOF mutations (n = 3), or one patient following hematopoietic stem cell transplant were labeled with mAbs against CD34, CD19, CD20, CD10, IgM, and CD27. Proportions of pro-B (CD19⁺CD34⁺CD10⁺CD20⁻IgM⁻), pre-BI (CD19⁺CD34⁻CD10⁺CD20⁻IgM⁻), pre-BII (CD19⁺CD34⁻CD10⁺CD20^dimIgM⁻), immature (CD19⁺CD34CD10⁺CD20⁺ IgM⁺), and recirculating mature (CD19⁺CD34⁻CD10⁻CD20⁺) B cells were determined. (A–C) Contour plots in show CD34 versus CD10 staining to identify pro-B cells, which were then further analyzed for pre-BI, pre-BII, immature, and recirculating mature according to differential expression of CD20 and CD10. (D) Mean ± SEM of the different subsets of B cells in the BM. (E) Representative contour plots showing CD10 versus CD27 staining on CD20⁺ B cells, and (F) mean ± SEM of CD10⁺CD27⁻, naive (CD20⁺CD10⁻CD27⁻) and memory (CD20⁺CD27⁺) B cells in the BM. Significant differences were determined by Student's t test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.