Surrogate light chains (psi L) encoded by lambda-like (lambda 5) and VpreB genes play a critical role in controlling the early steps of B cell differentiation. We prepared new anti-VpreB monoclonal antibodies (mAb) (3C7/6F6) which preferentially recognize the VpreB epitope at the cell surface of human cell lines that do not express the mu chain. These mAb provide the first characterization of human pro-B cell lines expressing surface psi L. We demonstrate that surface psi L expression is considerably enhanced upon interleukin-7 stimulation and that the psi L complex is formed independently of the Ig alpha/Ig beta heterodimer. Finally, using these antibodies, we confirm the existence of a normal pro-B cell population in human adult bone marrow. These cells are CD34+ CD38+ psi L+, do or do not express CD19, CD10, or both epitopes, and may represent the earliest cell population committed to B cell differentiation.