This study investigates the role of Piezo1 channels in vascular architecture and physiological force regulation. The research reveals that Piezo1 is a critical sensor for detecting frictional force (shear stress) and plays a pivotal role in determining vascular structure during development and in adult physiology. Global or endothelial-specific disruption of Piezo1 in mice leads to severe vascular defects and embryonic lethality, highlighting its essential function in vascular development. Haploinsufficiency of Piezo1 results in endothelial abnormalities in mature vessels, indicating its importance in maintaining vascular integrity. The study demonstrates that Piezo1 channels are essential for shear stress-evoked ionic currents and calcium influx in endothelial cells. The presence of Piezo1 enables endothelial cells to respond to shear stress, which is crucial for vascular development and maintaining a healthy vasculature. The data suggest that Piezo1 channels act as integrators in vascular biology, linking mechanical forces to cellular responses. Further analysis shows that Piezo1 depletion suppresses shear stress-evoked calcium entry and ionic currents in endothelial cells, indicating its role in sensing mechanical forces. The study also reveals that Piezo1 is sufficient to confer shear stress-evoked calcium entry, emphasizing its importance in endothelial cell function. The research highlights the functional significance of shear stress-activated Piezo1 channels in endothelial cell alignment and organization. Piezo1 channels are localized at the leading apical lamellipodia in response to shear stress, facilitating early-stage alignment of endothelial cells in the direction of flow. This process is crucial for vascular development and maintaining proper vascular architecture. The study also explores downstream mechanisms, showing that Piezo1 activity is linked to endothelial nitric oxide synthase (eNOS), which is essential for endothelial cell migration. The findings suggest that Piezo1 activity drives endothelial cell migration through eNOS in the absence of shear stress. Additionally, the study identifies calpain-2 as a downstream mechanism regulated by Piezo1. Calpain-2 is involved in the proteolytic cleavage of actin cytoskeletal and focal adhesion proteins, which is crucial for endothelial cell alignment and vascular development. The findings indicate that Piezo1-mediated calcium entry and downstream activation of calpain-2 are essential for coupling shear stress to endothelial cell organization and alignment. The study has important implications for understanding vascular physiology and potential disease processes such as atherosclerosis and cancer, where alterations in shear stress and other mechanical forces are common. The findings provide insights into the molecular mechanisms underlying vascular development and function, highlighting the critical role of Piezo1 channels in integrating mechanical forces with vascular architecture.This study investigates the role of Piezo1 channels in vascular architecture and physiological force regulation. The research reveals that Piezo1 is a critical sensor for detecting frictional force (shear stress) and plays a pivotal role in determining vascular structure during development and in adult physiology. Global or endothelial-specific disruption of Piezo1 in mice leads to severe vascular defects and embryonic lethality, highlighting its essential function in vascular development. Haploinsufficiency of Piezo1 results in endothelial abnormalities in mature vessels, indicating its importance in maintaining vascular integrity. The study demonstrates that Piezo1 channels are essential for shear stress-evoked ionic currents and calcium influx in endothelial cells. The presence of Piezo1 enables endothelial cells to respond to shear stress, which is crucial for vascular development and maintaining a healthy vasculature. The data suggest that Piezo1 channels act as integrators in vascular biology, linking mechanical forces to cellular responses. Further analysis shows that Piezo1 depletion suppresses shear stress-evoked calcium entry and ionic currents in endothelial cells, indicating its role in sensing mechanical forces. The study also reveals that Piezo1 is sufficient to confer shear stress-evoked calcium entry, emphasizing its importance in endothelial cell function. The research highlights the functional significance of shear stress-activated Piezo1 channels in endothelial cell alignment and organization. Piezo1 channels are localized at the leading apical lamellipodia in response to shear stress, facilitating early-stage alignment of endothelial cells in the direction of flow. This process is crucial for vascular development and maintaining proper vascular architecture. The study also explores downstream mechanisms, showing that Piezo1 activity is linked to endothelial nitric oxide synthase (eNOS), which is essential for endothelial cell migration. The findings suggest that Piezo1 activity drives endothelial cell migration through eNOS in the absence of shear stress. Additionally, the study identifies calpain-2 as a downstream mechanism regulated by Piezo1. Calpain-2 is involved in the proteolytic cleavage of actin cytoskeletal and focal adhesion proteins, which is crucial for endothelial cell alignment and vascular development. The findings indicate that Piezo1-mediated calcium entry and downstream activation of calpain-2 are essential for coupling shear stress to endothelial cell organization and alignment. The study has important implications for understanding vascular physiology and potential disease processes such as atherosclerosis and cancer, where alterations in shear stress and other mechanical forces are common. The findings provide insights into the molecular mechanisms underlying vascular development and function, highlighting the critical role of Piezo1 channels in integrating mechanical forces with vascular architecture.