The function of the cell surface molecule CD40 on B cell precursors (BCP) is not well understood. We now report studies using the L cell/CD40 system (anti-CD40 mAb immobilized on CD32+ mouse L cells) to assess the potential function of CD40 during human B cell ontogeny. Stimulation of human B lineage cells with IL-4 in the L cell/CD40 system yielded a hierarchy of responsiveness: high density tonsillar B cells > fetal splenic B cells > fetal bone marrow surface Ig+ immature B cells > fetal bone marrow surface Ig- BCP. Using a microsphere/flow cytometry growth quantitation assay, we found that substituting IL-3 for IL-4 in the L cell/CD40 system provided a stronger growth stimulus for fetal bone marrow BCP and immature B cells. We also found that FACS-purified fetal bone marrow CD10+/CD34+/CD40+/cytoplasmic mu- pro-B cells responded maximally to IL-3 plus IL-7. Surprisingly, anti-CD40 inhibited the pro-B cell response to IL-7. In contrast, FACS-purified fetal bone marrow CD10+/CD34-/CD40+/cytoplasmic mu+ pre-B cells were essentially nonresponsive to IL-3, IL-7, or anti-CD40 alone, but were uniquely responsive to IL-3 plus anti-CD40. B-lineage cells derived after 14 days from IL-7-stimulated pro-B cells were predominantly CD19+/L chain-, whereas pre-B cells stimulated with IL-3 plus anti-CD40 were predominantly CD19+/L chain+. The L chain+ cells from pre-B cell cultures were both mu+/delta+ and mu-/delta+. Our results demonstrate that the response to CD40 signaling depends upon the BCP developmental stage and the IL costimulus, and indicate that normal human pro-B cells and pre-B cells have different growth factor requirements.