In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3‐E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF‐α in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4–6 h of exposure to TNF‐α and CHX, and application of FSS (12 dyne/cm²) significantly attenuated this TNF‐α‐induced apoptosis. FSS activated PI3‐kinase signaling, induced phosphorylation of Akt, and inhibited TNF‐α‐induced activation of caspase‐3. Inhibition of PI3‐kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF‐α‐induced apoptosis and blocked FSS‐induced inhibition of caspase‐3 activation in osteoblasts treated with TNF‐α. LY294002 did not, however, prevent FSS‐induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3‐kinase‐independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF‐α‐induced apoptosis through a mechanism involving activation of PI3‐kinase signaling and inhibition of caspases. FSS‐induced activation of PI3‐kinase may promote cell survival through a mechanism that is distinct from the Akt‐mediated survival pathway. © 2002 Wiley‐Liss, Inc.