Formation of the pre-BCR complex is a critical check point during B cell development and induces the transition of pro-B to pre-B cells. CD79b (Igbeta) is a signaling component in the pre-BCR complex, since differentiation to the pre-B phenotype is induced by cross-linking the CD79b expressed on developmentally arrested pro-B cells from recombination-activating gene (RAG)-2-deficient mice. Bruton's tyrosine kinase (BTK) plays important roles in B cell development. However, its molecular mechanisms in early B cell development are not fully understood. To examine whether BTK functions in CD79b-mediated signaling for the pro-B/pre-B transition, we utilized RAG2/BTK double-knockout (DKO) mice. Pro-B cells from RAG2/BTK-DKO mice did not differentiate into pre-B cells following CD79b cross-linking, although tyrosine phosphorylation of cellular proteins including Erk1/2 and phospholipase C-gamma2 was induced in the same manner as RAG2-KO mice. BTK is phosphorylated after cross-linking of CD79b on RAG2-deficient pro-B cells. These findings suggest that BTK-dependent pathways downstream of CD79b are critical for the pro-B/pre-B transition and BTK-independent signaling pathways are also activated via the pre-BCR complex.