W hile efficient at treating B-cell malignancies, Bruton tyrosine kinase (BTK) inhibitors are consistently reported to increase the risk of bleeding. Analyzing platelet aggregation response to collagen in platelet-rich plasma allowed us to identify two groups in the healthy population characterized by low or high sensitivity to ibrutinib in vitro. Inhibition of drug efflux pumps induced a shift from ibrutinib low-sensitive platelets to high-sensitive ones. At a clinically relevant dose, acalabrutinib, a second-generation BTK inhibitor, did not affect maximal collagen-induced platelet aggregation in the ibrutinib low-sensitive group but did inhibit aggregation in a small fraction of the ibrutinib high-sensitive group. Consistently, acalabrutinib delayed aggregation, particularly in the ibrutinib high-sensitive group. In chronic lymphocytic leukemia patients, acalabrutinib inhibited maximal platelet aggregation only in the ibrutinib high-sensitive group. Acalabrutinib inhibited collagen-induced tyrosine-753 phosphorylation of phospholipase Cγ2 in both groups, but, in contrast to ibrutinib, did not affect Src-family kinases. Acalabrutinib affected thrombus growth under flow only in the ibrutinib high-sensitive group and potentiated the effect of cyclooxygenase and P2Y 12 receptor blockers in both groups. Since the better profile of acalabrutinib was observed mainly in the ibrutinib lowsensitive group, replacement therapy in patients may not systematically reduce the risk of bleeding.