Summary. Dithiothreitol (DTT) is known to induce an active conformation of αIIbβ3 integrin and to promote the aggregation of Chinese hamster ovary (CHO)-αIIbβ3 cells in the presence of soluble fibrinogen (Fg). The aim of this study was to compare adhesion and spreading with Fg or von Willebrand factor (VWF) of CHO-αIIbβ3 cells in the presence or absence of DTT. Our results indicate that DTT treatment was required to induce cell spreading on VWF. In contrast, CHO-αIIbβ3 cell spreading on Fg was already optimal in the absence of DTT. We used a small perfusion chamber coupled to videomicroscopy to demonstrate that CHO-αIIbβ3 cells that were adherent and spread on VWF required DTT activation to resist to detachment under increasing shear rates (50–1600/s). In contrast, untreated or DTT-treated cells spread on Fg were able to resist to extremely high flow rates. These data provide novel evidence that activated αIIbβ3 is absolutely required for spread cells to resist detachment and strengthens the importance of the αIIbβ3 activation step for adhesion and spreading to VWF.