To better understand the process of B-lymphocyte lineage restriction, we have investigated molecular and functional properties in early B-lineage cells from Pax-5-deficient animals crossed to a B-lineage-restricted reporter mouse, allowing us to identify B-lineage-specified progenitors independently of conventional surface markers. Pax-5 deficiency resulted in a dramatic increase in the frequency of specified progenitor B-cells marked by expression of a λ5 (Igll1) promoter-controlled reporter gene. Gene expression analysis of ex vivo isolated progenitor cells revealed that Pax-5 deficiency has a minor impact on B-cell specification. However, single-cell in vitro differentiation analysis of ex vivo isolated cells revealed that specified B-lineage progenitors still displayed a high degree of plasticity for development into NK or T lineage cells. In contrast, we were unable to detect any major changes in myeloid lineage potential in specified Pax-5-deficient cells. By comparison of gene expression patterns in ex vivo isolated Pax-5- and Ebf-1-deficient progenitors, it was possible to identify a set of B-cell-restricted genes dependent on Ebf-1 but not Pax-5, supporting the idea that B-cell specification and commitment is controlled by distinct regulatory networks.