The expression of B lineage associated genes during early B cell differentiation stages is not firmly established. Using cell surface markers and multiparameter flow cytometry, bone marrow (BM) cells can be resolved into six fractions, representing sequential stages of development; i.e., pre-Pro-B, early Pro-B, late Pro-B/large Pre-B, small Pre-B, immature B, and mature B cells. Here we quantitate the levels of several B lineage associated genes in each of these fractions by RT-PCR, demonstrating different patterns of expression. We find that expression of terminal deoxynucleotidyl transferase (TdT), lambda 5, and VpreB is predominantly restricted to the Pro-B stages. Rag-1 and Rag-2 expression is also tightly regulated, and is found largely in the Pro-B through small Pre-B stages. Mb-1 is present from Pro-B throughout the pathway at high levels. Finally, Bcl-2 is expressed at high levels only at the pre-Pro-B and mature B stages, whereas it is low during all the intermediate stages. We also correlate this expression data with an analysis of the onset of Ig gene rearrangement as assessed by amplifying D-JH, VH-DJH, and VK-JK. Finally, we report differences in gene expression during B lymphopoiesis at two distinct ontogenic timings, in fetal liver and adult BM: both TdT and the precursor lymphocyte regulated myosin-like light chain are expressed at high levels in the Pro-B cell stage in bone marrow, but are absent from the corresponding fraction in fetal liver. In contrast, lambda 5, VpreB, Rag-1, and Rag-2 are expressed at comparable levels.