Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its subunit's cytoplasmic domain connected to the cytoskeleton. This culminates in signals directing cellular responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin IIb3 is continuously subjected to frictional shearing forces generated by blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on IIb3, we examined IIb3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that -actinin, myosin heavy chain and Syk co-immunoprecipitate with IIb3 in resting platelets, and that 120 dynes/cm² shear stress leads to their disassociation from IIb3. Shear-induced disassociation of -actinin and myosin heavy chain from the 3 tail is unaffected by blocking VWF binding to GpIb-IX-V but abolished by blocking VWF binding to IIb3. Syk's disassociation from 3 is inhibited when VWF binding to either GpIb-IX-V or IIb3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine phosphorylated ADAP are inhibited by blocking IIb3 but not by blocking GpIb-IX-V. CHO cells expressing IIb3 with 3 truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress modulates IIb3 function, and suggest that shear-induced IIb3-mediated signaling contributes to the regulation of platelet aggregation by directing the release of cytoskeletal elements from the 3 tail.