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The Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. In this study, we demonstrated that changes in cell morphology are a key factor. Morphological manipulation of single cells without cell-cell contact resulted in flat spread or round compact cells with nuclear or cytoplasmic Yap , respectively. Stress fibers increased in response to expanded cell areas, and F-actin regulated Yap downstream of cell morphology. Cell morphology- and F-actin-regulated phosphorylation of Yap , and the effects of F-actin were suppressed by modulation of Lats. Our results suggest that cell morphology is an important factor in the regulation of the Hippo pathway, which is mediated by stress fibers consisting of F-actin acting upstream of, or on Lats, and that cells can detect density through their resulting morphology (Figure). This cell morphology (stress-fiber)-mediated mechanism probably cooperates with a cell-cell contact (adhesion)-mediated mechanism involving the Hippo pathway to achieve density-dependent control of cell proliferation.