Previous studies in Chinese hamster ovary cells showed that truncational mutations of β3 at sites of F754 and Y759 mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F754 to C-terminus and the conservative N756ITY759 motif in platelet function have yet to be elaborated. Mice expressing β3 with F754 and Y759 truncations, or NITY deletion (β3-ΔTNITYRGT, β3-ΔRGT, or β3-ΔNITY) were established through transplanting the homozygous β3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different β3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the β3-ΔTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the β3-ΔNITY platelets. Behaviors of the β3-ΔRGT platelets were consistent with the previous observations in the β3-ΔRGT knock-in platelets. The adhesion impairment of platelets with the β3 mutants under flow was in different orders of magnitude shown as: β3-ΔTNITYRGT>β3-ΔRGT>β3-ΔNITY to fibrinogen-coated surface, and β3-ΔTNITYRGT>β3-ΔNITY>β3-ΔRGT to collagen-coated surface. To evaluate the interaction of the β3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that β3-ΔRGT interacted with kindlin but not c-Src, β3-ΔNITY interacted with c-Src but not kindlin, while β3-ΔTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin β3, i.e. T755NITYRGT762, R760GT762, and N756ITY759 participate in bidirectional, outside-in, and inside-out signaling, respectively and the association of c-Src or kindlin with β3 integrin may regulate these processes.