The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis [1,2,3]. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment, depletion (methyl-β-Cyclodextrin; MBCD) and the disruption of membrane cholesterol organization (Dynasore) affects PIEZO1's response to mechanical force. STORM super-resolution imaging revealed that, at the nano-scale, PIEZO1 channels in the membrane associate as clusters which increase in number upon cholesterol addition. Both cluster size and diffusion rates were profoundly affected by treatment with MBCD (5 mM). In addition, electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1 pressure-response curve and increased channel latency in response to mechanical stimuli. Our results indicate that PIEZO1 function is directly dependent on the amount and lateral organization of membrane cholesterol which is crucial for the concerted inactivation of PIEZO1 clusters. 1 Cahalan SM, et al. Piezo1 links mechanical forces to red blood cell volume. eLife. 2015;4. 2 Rode B, et al. Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance. Nat Commun. 2017;8. 3 Li J, et al. Piezo1 integration of vascular architecture with physiological force. Nature, 2014. Supported by the Australian Research Council and the National Health and Medical Research Council of Australia.