The 75-kD HS1 protein is highly tyrosine-phosphorylated during B cell antigen receptor (BCR)-mediated signaling. Owing to low expression of HS1, WEHI-231-derived M1 cells, unlike the parental cells, are insensitive to BCR-mediated apoptosis. Here, we show that BCR-associated tyrosine kinases Lyn and Syk synergistically phosphorylate HS1, and that Tyr378 and Tyr-397 of HS1 are the critical residues for its BCR-induced phosphorylation. In addition, unlike wild-type HS1, a mutant HS1 carrying the mutations Phe-378 and Phe-397 was unable to render M1 cells sensitive to apoptosis. Wild-type HS1, but not the mutant, localized to the nucleus under the synergy of Lyn and Syk. Thus, tyrosine phosphorylation of HS1 is required for BCR-induced apoptosis and nuclear translocation of HS1 may be a prerequisite for B cell apoptosis.