Abstract Commitment of hematopoietic progenitor cells to B-lymphoid cell fate has been suggested to coincide with the development of PAX5-expressing B220+CD19+ pro–B cells. We have used a transgenic reporter mouse, expressing human CD25 under the control of the B-lineage–restricted Igll1 (λ5) promoter to investigate the lineage potential of early progenitor cells in the bone marrow. This strategy allowed us to identify a reporter expressing LIN−B220−CD19−CD127+FLT3+SCA1lowKITlow population that displays a lack of myeloid and a 90% reduction in in vitro T-cell potential compared with its reporter-negative counterpart. Gene expression analysis demonstrated that these lineage-restricted cells express B-lineage–associated genes to levels comparable with that observed in pro–B cells. These data suggest that B-lineage commitment can occur before the expression of B220 and CD19.