Analysis of primitive progenitors in Hmgb3^–/Y mice. (A) Representative isolation of wild-type common lymphoid and myeloid progenitors by flow cytometry. (Ai) Lin^– bone marrow isolated from wild-type mice. (Aii) CLP and HSC populations were isolated from c-kit and Sca-1 double-positive lin^– bone marrow cells. Double-positive cells were further segregated into c-kit^LO, Sca-1^LO (solid box) and c-kit^HI, Sca-1^HI (dotted box) populations. Regions were drawn based on isotype staining of littermate control cells. (Aiii) The CMP population was isolated from c-kit⁺, Sca-1^–, and IL-7Rα^– lin^– bone marrow cells. (Aiv) Further isolation of the HSC population based on IL-7Rα expression in c-kit^HI, Sca-1^HI cells. Approximately 90% of c-kit^LO, Sca-1^LO cells were IL-7Rα^– (HSC phenotype). (Av) Isolation of the CLP population based on IL-7Rα expression in c-kit^LO, Sca-1^LO cells. Approximately 89% of c-kit^LO, Sca-1^LO cells were IL-7Rα⁺ (CLP phenotype). (Avi) Isolation of the CMP population based on FcγRII/III and CD34 expression in c-kit⁺, Sca-1^–, and IL-7Rα^– cells. Cells that are FcγRII/III^LO and CD34⁺ represent the CMP phenotype. (B) Average number of HSCs (lin^–, c-kit^HI, Sca-1^HI, IL-7Rα^–; n = 3), CLPs (lin^–, c-kit^LO, Sca-1^LO, IL-7Rα⁺; n = 3), and CMPs (lin^–, c-kit⁺, Sca-1^–, IL-7Rα^–, FcγRII/III^LO, CD34⁺; n = 4) as determined by the percentage of lineage-negative (lin^–) bone marrow cells that stained positive for the HSC, CLP, or CMP phenotype. Staining was performed on lineage-depleted cells isolated from groups of 3 Hmgb3^–/Y or wild-type mice prior to analysis by flow cytometry. The data represent the pooled results of 3 (HSCs and CLPs) or 4 (CMPs) independent experiments. (C) Mean CFU–Pre-B frequency generated from wild-type and Hmgb3^–/Y CLP populations. CFU–Pre-B frequency within the CLP population was determined by scoring pre–B-cell colonies per 1000 cells cultured: wild-type (n = 8; 355 CFU–Pre-Bs counted); Hmgb3^–/Y (n = 8; 890 CFU–Pre-Bs counted). The data represent the pooled results of 2 independent experiments. (D) Mean myeloid colony (CFU-C) frequency generated from wild-type and Hmgb3^–/Y CMP populations. Myeloid CFU-C frequency within the CMP population was determined by scoring CFU-GM, BFU-E, and CFU-GEMM colonies per 500 cells cultured: wild-type (n = 10; 813 CFU-GMs, 177 BFU-Es, and 91 CFU-GEMMs counted); Hmgb3^–/Y (n = 10; 239 CFU-GMs, 47 BFU-Es, and 17 CFU-GEMMs counted). The data represent the pooled results of 2 independent experiments. P values in panels B-D were determined by Student t test. In panels B-D, error bars represent standard deviation.