IL-7 plays a critical role in B cell fate decision by regulating early B cell factor (EBF) expression. However, it was not clear when IL-7 stimulation is necessary in hemato-/lymphopoiesis in adult mice. Here we show that pre-proB cells derived from IL-7−/− mice have lost B cell potential, despite up-regulation of EBF expression following IL-7 stimulation. Pre-proB cells from wild-type mice can give rise to proB cells in the absence of IL-7. In this case, EBF up-regulation during the transition from the pre-proB to proB stages occurs normally. In contrast, EBF expression by IL-7−/− pre-proB cells after IL-7 stimulation is∼ 20 times lower than wild-type pre-proB cells. In addition, only multipotent progenitors with higher levels of ectopic EBF can give rise to proB cells in the absence of IL-7. Therefore, the primary function of IL-7 before the pre-proB stage in B cell development is to maintain the EBF expression level above a certain threshold, which is necessary for pre-proB cells to further transit to the proB stage.