Loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. However, the nature of the stimulus that activates the cell is debated. Candidate stimuli include wall shear stress, streaming potentials, and chemotransport. We have addressed the nature of the flow-derived cell stimulus by comparing variations in fluid transport with variations in wall shear stress, using nitric oxide (NO) and prostaglandin E₂ (PGE₂) production as a parameter of bone cell activation. Adult mouse long bone cell cultures were treated for 15 min with or without pulsating fluid flow using the following regimes:​ Low PFF, mean flow rate 0.20 cm³/s, 3 Hz, shear stress 0.4±0.12 Pa;​ Medium PFF, 0.33 cm³/s, 5 Hz, 0.6±0.27 Pa;​ and High PFF, 0.63 cm³/s, 9 Hz, 1.2±0.37 Pa. In some Low PFF experiments, 2.8% neutral dextran (mol. wt. 4.98×10⁴) was added to the flow medium to increase the viscosity, thereby increasing the wall shear stress 3-fold to a level similar of the High PFF stimulus, but without affecting streaming potentials or chemotransport. NO and PGE₂ production were stimulated by Low, Medium, and High PFF in a dose-dependent manner. Application of Low PFF using dextran-supplemented medium, enhanced both the NO and PGE₂ response by 3-fold, to a level mimicking the response to High PFF at normal viscosity. These results show that the production of NO and PGE₂ by bone cells can be enhanced in a dose-dependent manner by fluid flow of increasing wall shear stress. Therefore, the stimulus leading to NO and PGE₂ production is the flow-derived shear stress, and not streaming potentials or chemotransport.