Clonable B cells arose in liquid cultures of slg+ cell-depleted bone marrow suspensions, and a majority of these derived from rapidly sedimenting (presumably large) precursors. Such immature (pre-B) cells were not demonstrable in adult lymph nodes. The development of functional B cells in vitro depended on adherent cells present in adult bone marrow or peritoneal exudates and in the presence of optimal numbers of these accessory cells; not FCS or 2-ME or LPS were essential for B cell maturation. In contrast, the ability of fetal liver cell suspensions to generate B cells in culture was not affected by the removal of adherent cells or addition of macrophages, and LPS consistently reduced numbers of colony-forming B cells recovered. A type of nonadherent cell present in the bone marrow of adult CBA/N mice dramatically enhanced B cell maturation in normal CBA/H-T6T6 fetal liver cell cultures. These results indicate that 2 types of accessory cells may augment transition of pre-B cells to functional B cells in culture. One is adherent and can be replaced by macrophages, whereas the other is nonadherent and is present in limiting numbers in embryos. When fetal liver cultures were incubated with optimal numbers of adult accessory cells, the rate of emergence of B cells was dependent on the gestational age of the donor embryo. This suggests that B cell formation in utero may be limited by the availability of B cell precursors as well as nonadherent accessory cells. The possibility exists that fetal pre-B cells typify intermediates in the B lineage which are infrequent in adult marrow. Alternatively, B cells may be produced in adult life through processes that do not precisely recapitulate embryonic events.