Fetal BM CD10^– PreProB-progenitors lie upstream of CD10⁺ ProB-progenitors in the fetal B-cell developmental hierarchy. (A) B-cell differentiation assay of flow-sorted PreProB-progenitors and ProB-progenitors cocultured on fetal BM MSCs with cytokines (FLT3L, SCF, IL-7, and IL-2) showing proportions of progenitors and mature B-cells produced by days 3, 7, and 14 (results expressed as mean - SEM, n = 3). (B) Pattern of IgH gene rearrangement detected in whole-genome amplified DNA from flow-sorted fetal BM HSPCs expressed as percentage of samples tested (n = 3-7 for each sub-population). (C) Diffusion map of single-cell qRT-PCR (total of 938 cells from 2 biological samples) of index-sorted Lin2⁻CD34⁺ or Lin2⁻CD34^–CD19⁺ fetal BM cells showing B-lymphoid and myeloid/erythroid differentiation trajectories. Genes and TaqMan probes detailed in supplemental Table 1. (D) Diffusion map of single-cell qRT-PCR (total of 1400 cells from 3 biological samples) of flow-sorted fetal BM HSPCs and B cells showing differentiation trajectory from HSCs through to B cells. Genes and TaqMan probes are detailed in supplemental Table 2. (E) Normalized gene expression of selected B cell–associated genes in flow-sorted fetal BM HSPCs by single-cell qRT-PCR (total of 1400 cells from 3 biological samples) .