Environmental factors essential for the first stages of B lymphopoiesis remain elusive. Here, we report that immediately after commitment to B lineage, precursors become dependent on a chemokine SDF-1 and its receptor CXCR4 using mutant and radiation chimeric mice. In bone marrow, generation of the earliest identifiable B cell precursor populations requires CXCR4. In fetal liver, we identified Lin⁻CD19⁻c-kit⁺IL-7Rα⁺AA4.1⁺, the earliest unipotent B cell precursor population, and found that its development was severely affected in SDF-1^−/− embryos but not in IL-7^−/− embryos. Lin⁻ T cell progenitors appeared normal in SDF-1^−/− embryos. Moreover, SDF-1 exhibited specific biologic activities on the earliest B cell precursors. SDF-1 provides the first example of a cytokine responsible for the earliest B lineage stages.