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B-cell receptor (BCR) signaling is essential for normal B cell development and proliferation. In addition, functional BCR signaling contributes to the proliferation and survival of many B cell lymphomas through constitutive low level or tonic signaling. Disruption of this tonic signaling pathway results in cell death or decreased viability in many B cell lymphoma cells. Bruton’s tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases and plays a crucial role in the development and activation of B cells through association with the BCR signalosome. We have developed a novel, potent, irreversible inhibitor of Btk that selectively disrupts the BCR signaling pathway as measured by inhibition of both Btk Y223 autophosphorylation and phosphorylation of the Btk substrate PLCg2. AVL101 inhibits Btk but leaves upstream BCR kinase activity intact; both Syk phosphorylation at Y525/526 and transphosphorylation of Btk on residue Y551 are unaltered. By mass spectrometry, we have shown that AVL101 covalently modifies Btk specifically at Cys-481, and in cell-based assays, this irreversible binding leads to prolonged inhibition of Btk kinase activity. After removal of the compound, inhibition of Btk activity is maintained in cultured B cell lymphoma cells for > 8 hours. This prolonged inhibition correlates with the half-life of the Btk protein, which we have found to be greater than 8 hours, irrespective of BCR activation state or modification by the inhibitor. We have demonstrated that AVL101 functionally inhibits BCR signaling in vitro and in vivo. In vitro, AVL101 inhibits BCR ligand-induced calcium flux as well as proliferation of the B cell lymphoma cell lines DOHH2 and WSU-DLCL2. In vivo, we have shown AVL101 is orally bioavailable and can inhibit Btk-dependent B cell function. Our data demonstrate that Btk is a valid therapeutic target for B cell lymphomas and other disorders dependent on BCR signaling. Furthermore, irreversible inhibition of Btk enables complete and prolonged inactivation of the target kinase, and suggests that clinically, a rapid and sustained therapeutic response may be possible using an orally available, irreversible Btk inhibitor.