The capacity of precursor B (pre-B) I cells from fetal liver and bone marrow to proliferate and differentiate into surface immunoglobulin-positive immature B cells in vitro was analyzed. Both fetal liver- and bone marrow-derived progenitors do so in a pre-B cell receptor (pre-BCR)-dependent manner in tissue culture medium alone, without addition of other cells or cytokines. Approximately 20% of the initial pre-B I cells enter more than one division. Analyses at the single-cell level show that approximately 15% divide two to five times. Coculture of pre-B I cells with stromal cells did not enhance proliferation or differentiation, whereas the presence of interleukin 7, especially in combination with stromal cells, resulted mainly in the expansion of pre-B I cells and prevented their further differentiation. Thus, the environment of fetal liver or bone marrow is not required for the pre-BCR to exert its function, which is to select and expand cells that have undergone an inframe V(H)-D(H)J(H) rearrangement that produces a pre-BCR-compatible muH chain. It appears unlikely that a ligand for the pre-BCR drives this pre-B cell proliferation.