Studies here respond to two long-standing questions: Are human "pre/pro-B" CD34 + CD10 − CD19 + and "common lymphoid progenitor (CLP)/early-B" CD34 + CD10 + CD19 − alternate precursors to "pro-B" CD34 + CD19 + CD10 + cells, and do the pro-B cells that arise from these progenitors belong to the same or distinct B-cell development pathways? Using flow cytometry, gene expression profiling, and Ig V H -D-J H sequencing, we monitor the initial 10 generations of development of sorted cord blood CD34 high Lineage − pluripotential progenitors growing in bone marrow S17 stroma cocultures. We show that (i) multipotent progenitors (CD34 + CD45RA + CD10 − CD19 − ) directly generate an initial wave of Pax5 + TdT − "unilineage" pre/ pro-B cells and a later wave of "multilineage" CLP/early-B cells and (ii) the cells generated in these successive stages act as precursors for distinct pro-B cells through two independent layered pathways. Studies by others have tracked the origin of B-lineage leukemias in elderly mice to the mouse B-1a pre/pro-B lineage, which lacks the TdT activity that diversifies the V H -D-J H Ig heavy chain joints found in the early-B or B-2 lineage. Here, we show a similar divergence in human B-cell development pathways between the Pax5 + TdT − pre/ pro-B differentiation pathway that gives rise to infant B-lineage leukemias and the early-B pathway. Ordered stages in the current human model are termed the "common lymphoid progenitor" (CLP) and the "early-B," "progenitor-B," and "precursor-B" subsets that follow it (i.e., SC → CLP → early-B → pro-B → pre-B → B) (5). In this pathway, CLP and early-B stages, which both express the CD34 + CD45RA + CD10 + CD19 − surface phenotype, are actually "multilineage" progenitors of B, T, dendritic (DC), and natural killer (NK) cells (5, 10-12).Pro-B cells, which retain CD34 and CD10 expression but also express CD19 (CD34 + CD10 + CD19 + ), constitute the first Pax-5 + committed B-lineage stage, which is followed by pre-B (CD34 − CD10 + CD19 + ) cells that have ceased expression of CD34 and now express the surface μH-VpreB-λ5/CD79 surrogate Ig receptor complex (1-5, 13-15). Development of the latter CD34 − pre-B cells and their B-cell and Ig-secreting plasma cell progeny can be reconstituted after coculture of purified CD34 + Lineage − (Lin − ) progenitors with bone marrow (BM) stroma lines (e.g., S17) (15-18).Before the CLP subset and the above developmental pathway were recognized, a subset called "pre/pro-B," which expresses the CD34 + CD10 − CD19 + surface phenotype, was commonly considered to be the first human B-lineage stage (19). It occurs in fetal liver, fetal bone marrow (FBM), and umbilical cord blood (CB) (19-21). An FBM pre/pro-B cell stage was shown to distinctively lack TdT and to bear CD7 unlike concurrent conventional CD10 + CD19 − B-cell progenitors, leading to the idea that these cells might belong to a distinct B lineage (20). This lack of TdT-mediated "N-nucleotide additions" between D and J H segments is further associated with differential V-D-J gene...