Mechanosensation is essential for life. At the frontline of rapid mechanosensation in mammals are the Piezo ion channels, and most widely Piezo1. Since its discovery, Piezo1 has been identified as a ubiquitous mechanosensor, transducing diverse types of mechanical stimuli in processes from touch to blood flow and organ formation. However, how cells sense and respond to different types of mechanical force through Piezo1 is not well understood, in part because we lack a molecular description of the channel’s forcedependent activation mechanism. Here, I show how vesicles derived from cells retain pressure-dependent ion channel activity of Piezo1, in contrast to previously used reconstitution methods. Single-particle cryoelectron microscopy analysis of Piezo1 in cell-derived vesicles reveals a fully flattened conformation under force, with flattening coupled to pore expansion and rearrangements of the gating apparatus necessary for channel activation. Analysis of Piezo1 conformations from differing vesicle sizes and comparisons to reconstituted structures describes a new switch-like force-dependent activation mechanism for Piezo1. This work provides a molecular framework from which we can now begin to probe the mechanisms of Piezo1 mechanosensation in different force-sensing processes and disease states.