Cells migrate in a directional manner in a variety of conditions, including chemoattractant gradients, electric fields, or under the influence of shear flow. Chemotaxis, or directed migration up a chemical gradient, is the best understood of these processes, and involves activation of multiple parallel signal transduction pathways that transmit the input from the chemoattractant receptor to the cytoskeletal network, leading to biased pseudopod projection in the direction of the gradient. Many components of the chemotactic signaling network display a polarized distribution in migrating cells. Although some of these markers have also been observed in cells migrating in an electric field or under shear flow, it is unclear whether these processes involve activation of a similar signaling network, or how they trigger cell migration. We found that acute mechanical stimulation of Dictyostelium cells leads to phosphorylation and activation of multiple components of the chemotactic signaling network, including PKB and ERK. Furthermore, using a microfluidic device, we demonstrated that application of shear flow for two seconds triggers translocation of leading and lagging edge markers to and from the cortex, respectively, similarly to global stimulation with a chemoattractant. Remarkably, the signaling network activated by acute mechanical stimulation displayed many behaviors characteristic of the chemotactic signaling network, including a refractory period, which is indicative of the system's excitatory nature. Simultaneous inhibition of multiple signaling pathways, including PI3K, PLA2 and TORC2, did not block the response to acute mechanical stimulation. In contrast, the response depended on the presence of an intact actin cytoskeleton, as well as Ca2+ flux mediated by the IP3 receptor homolog. Overall, these findings provide insights into the mechanism of shear stress induced migration, as well as a novel approach for studying the properties of the chemotactic signaling network without input from a chemoattractant.