Although pro-B cells are well represented in IL-7 knockout (KO) mice, they express abnormally low concentrations of the interleukin-7 receptor α-chain (IL-7Rα) and do not generate pre-B cells. Here, we demonstrate that pro-B cells from IL-7 KO mice can be induced to generate pre-B cells and immature B cells by exposure to recombinant IL-7 (rIL-7) in vivo but not in vitro. Experiments in recombinant activation gene-1 (RAG-1) KO mice indicate that the in vitro unresponsiveness of IL-7^-/- pro-B cells to rIL-7 is unrelated to the absence of a functional pre-B cell receptor (pre-BCR). Rather, it appears to be due to the suboptimal expression of the IL-7Rα chain. Thus, IL-7^-/- pro-B cells readily respond to rIL-7 in vitro if IL-7Rα chain expression is first upregulated by exposure to IL-7 in vivo or to IL-7^+/+ bone marrow (BM) stromal cells or conditioned medium (CM) therefrom in vitro. Similar results were obtained when pro-B cells from IL-7 KO mice were cultured on IL-7^-/- BM stromal cells in the presence of rIL-7. This suggested that the recently described pre-pro-B cell growth-stimulating factor (PPBSF), a self-assembling hybrid cytokine comprising IL-7 and the stromal cell-derived hepatocyte growth factor β-chain (HGFβ), is required to stimulate pro-B cells from IL-7 KO mice. This inference was verified by demonstrating that purified PPBSF upregulates IL-7Rα chain expression on IL-7^-/- pro-B cells in vitro and enables them to respond to rIL-7 in a stepwise manner. We, therefore, postulate that PPBSF is the operative form of IL-7 that normally induces IL-7Rα^lo pre-pro-B cells to proliferate and differentiate into IL-7Rα^hi pro-B cells, which then proliferate and differentiate into pre-B cells on stimulation with monomeric IL-7.