We found that the interaction of platelets with immobilized von Willebrand factor (VWF) under flow induces distinct elevations of cytosolic Ca⁺⁺ concentration ([Ca⁺⁺]_i) that are associated with sequential stages of integrin α_IIbβ₃ activation. Fluid-dynamic conditions that are compatible with the existence of tensile stress on the bonds between glycoprotein Ibα (GPIbα) and the VWF A1 domain led to Ca⁺⁺ release from intracellular stores (type α/β peaks), which preceded stationary platelet adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate, as well as membrane-permeable calcium chelators, inhibited these [Ca⁺⁺]_ioscillations and prevented stable adhesion without affecting the dynamic characteristics of the typical platelet translocation on VWF mediated by GPIbα. Once adhesion was established through the integrin α_IIbβ₃, new [Ca⁺⁺]_i oscillations (type γ) of greater amplitude and duration, and involving a transmembrane ion flux, developed in association with the recruitment of additional platelets into aggregates. Degradation of released adenosine diphosphate (ADP) to AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K) prevented this response without affecting stationary adhesion and blocked aggregation. These findings indicate that an initial signal induced by stressed GPIbα-VWF bonds leads to α_IIbβ₃ activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied α_IIbβ₃, with the contribution of a pathway involving PI3-K, amplify platelet activation to the level required for aggregation. Our conclusions modify those proposed by others regarding the mechanisms that regulate signaling between GPIbα and α_IIbβ₃ and lead to platelet adhesion and aggregation on immobilized VWF.