This study sought to assess the role of several signaling pathways in the fluid flow shear stress-induced proliferation and differentiation of normal human osteoblasts. We evaluated the effects of an effective dose of selective inhibitors of the extracellular signal-regulated kinases (ERK) pathway (PD98059 and U0126), the nitric oxide synthase pathway (N_ω-nitro-l-arginine methyl ester), the cyclo-oxygenase pathway (indomethacin), or the Gi/o pathway (pertussis toxin [PTX]) on the flow-mediated effects. A 30-min steady flow shear stress at 20 dynes/cm² increased significantly [3H]thymidine incorporation (an indicator of proliferation), alkaline phosphatase activity (an index of osteoblast differentiation), phosphorylation of ERK, and expression of integrin β1. PD98059, U0126, and Nω-nitro-l-arginine methyl ester completely blocked the shear stress-induced increases in ERK phosphorylation, [³H]thymidine incorporation, and alkaline phosphatase, but without an effect on integrin β1 expression, indicating that the ERK and nitric oxide synthase pathways are essential for the shear stress-induced proliferation and differentiation of normal human osteoblasts and that each involves ERK activation but not integrin β1 upregulation. Indomethacin blocked the shear stress-induced osteoblast proliferation and differentiation and integrin β1 upregulation but not ERK activation, suggesting that the cyclo-oxygenase pathway (i.e., prostacyclin and/or prostaglandin E2) mediates the shear stress-induced osteoblast proliferation in an ERK-independent manner. In contrast, PTX completely blocked the flow-induced increase in integrin β1 expression but had no effect on the increase in the ERK phosphorylation or [³H]thymidine incorporation. PTX not only did not inhibit but also significantly enhanced the stimulatory effect of shear stress on alkaline phosphatase activity, suggesting that a PTX-sensitive signaling pathway may have an inhibitory role in osteoblast differentiation. In summary, this study shows, for the first time, that the signal transduction mechanism of shear stress in osteoblasts is complex and involves multiple ERK-dependent and independent pathways, and provides circumstantial evidence that there may be a PTX-sensitive pathway that has completing effects with an unknown pathway on the differentiation of normal human osteoblasts.