Osteoarthritis is a chronic disease that can be initiated by altered joint loading or injury of the cartilage. The mechanically sensitive PIEZO ion channels have been shown to transduce injurious levels of biomechanical strain in articular chondrocytes and mediate cell death. However, the mechanisms of channel gating in response to high cellular deformation and the strain thresholds for activating PIEZO channels remain unclear. We coupled studies of single-cell compression using atomic force microscopy (AFM) with finite element modeling (FEM) to identify the biophysical mechanisms of PIEZO-mediated calcium (Ca ²⁺ ) signaling in chondrocytes. We showed that PIEZO1 and PIEZO2 are needed for initiating Ca ²⁺ signaling at moderately high levels of cellular deformation, but at the highest strains, PIEZO1 functions independently of PIEZO2. Biophysical factors that increase apparent chondrocyte membrane tension, including hypoosmotic prestrain, high compression magnitudes, and low deformation rates, also increased PIEZO1-driven Ca ²⁺ signaling. Combined AFM/FEM studies showed that 50% of chondrocytes exhibit Ca ²⁺ signaling at 80 to 85% nominal cell compression, corresponding to a threshold of apparent membrane finite principal strain of E = 1.31, which represents a membrane stretch ratio (λ) of 1.9. Both intracellular and extracellular Ca ²⁺ are necessary for the PIEZO1-mediated Ca ²⁺ signaling response to compression. Our results suggest that PIEZO1-induced signaling drives chondrocyte mechanical injury due to high membrane tension, and this threshold can be altered by factors that influence membrane prestress, such as cartilage hypoosmolarity, secondary to proteoglycan loss. These findings suggest that modulating PIEZO1 activation or downstream signaling may offer avenues for the prevention or treatment of osteoarthritis.