Lyn, Syk, SLP-76, and PI3K are required for Btk phosphorylation (Tyr223) in platelets stimulated with bt/VWF. Btk (Tyr223) was not phosphorylated in Lyn^-/- (A), Syk^-/- (B), and SLP-76^-/- (C) platelets in response to bt/VWF; the PI3K inhibitor wortmannin (100 nM) also blocked Btk (Tyr223) phosphorylation (D). Syk and SLP-76 were phosphorylated in Btk^xid/Y platelets stimulated by bt/VWF (E). Phosphorylation of Syk, SLP-76, and PLCγ2 was diminished in the presence of EDTA (5 mM); indomethacin (75 μM) treatment had no greater effect on the phosphorylation of Syk, SLP-76, and PLCγ2 than EDTA alone (F). EDTA also had no effect on the phosphorylation of Syk and SLP-76 in Btk^xid platelets (G). These results demonstrate that the activation of Btk in response to bt/VWF stimulation of platelets requires the functions of Lyn, Syk, SLP-76, and PI3K.