A simple one-step isolation technique significantly enriched mouse fetal liver cells that respond to interleukin 3 (IL-3), a multilineage hematopoietic growth factor. The fetal liver cell subpopulation isolated with monoclonal antibody AA4 contained 50- to 100-fold higher frequencies of multipotential (CFU-mix) or restricted (CFU-G/M, BFU-E) erythroid/myeloid precursors as well as precursors that differentiate to become mature B lymphocytes [CFU-mix = erythroid and myeloid colony-forming unit(s); CFU-G/M = CFU-granulocyte/macrophage; BFU-E = burst-forming unit-erythroid]. The B-lymphocyte precursors could be cloned in single-cell cultures when IL-3-containing supernatants were present. Growth of these clones was supported by purified IL-3 but not by purified IL-2. Stable growth has been maintained for greater than 6 mo in the presence of IL-3. Such clones express on their cell surface low amounts of class I major histocompatibility complex antigens and high amounts of AA4, GF1, and leukocyte common glycoprotein 200 antigens. They lack detectable rearrangements of their Ig-encoding genes [joining region heavy and light (kappa, lambda) chain genes], even after subcloning, but maintain their capacity to differentiate to mature B lymphocytes committed to multiple Ig specificities.