To get GUVs with phase-separated membranes, we chose the ternary lipid system composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) and cholesterol, which is known to phase-separate in liquid-ordered (Lo) and liquid-disordered domains (Ld) at room temperature for a range of specific compositions. The confinement was first achieved in parallelly arranged straight microfluidic channels enabling the capture of multiple GUVs in different lanes simultaneously (Figure , Figure ). GUVs with a typical diameter around 60 µm were confined in channels with a smaller square-shaped constriction (h = w = 20, 30, or 40 µm, where h and w denote the designed channel height and width, respectively; see Table for actual values). It allowed us to confine and deform GUVs into rod-shaped morphologies (Figure , middle) where the middle part of each confined GUV was in contact with the flat microchannel walls. In contrast, its extremities constituted free curved regions where the localization, growth and coalescence of lipid domains would thus be favored. GUVs were produced by electro-formation and introduced in the microfluidic device previously coated with-casein to limit membrane adhesion. The device was heated to 50 °C, i.e., above the mixing temperature (Tmix), to obtain an isotropic membrane and render GUVs more flexible by the increase of membrane surface area as well as by the decrease of their bending rigidity. Applying a small pressure allowed us to introduce the isotropic GUVs in the confinement channels (Figure ). Once the GUVs were confined, the flow inside channels was stopped (Figure ) to avoid any hydrodynamic effects on domain formation and redistribution. The system was then let to cool down to room temperature (Figure ) and below Tmix, triggering phase separation in the confined GUVs (Figure ). The domain distribution was observed by fluorescence microscopy (Figure ) using a small fraction (0.1 mol%) of fluorescentlylabeled phospholipid Rhodamine-DHPE, which specifically accumulated in the Ld phase (displayed in red), and NBD-PE, which distributed in both phases thus allowing to visualize the Lo phase in a separate fluorescence channel (displayed in blue). We first used a membrane composition DPPC/DOPC/Chol = 45/25/30 mol% leading to a Lo-rich membrane coexisting with a Ld phase in minority. Notably, time-lapse observation revealed that the Ld domains progressively accumulated at the free curved region and fused to decrease line tension, first at the "corners" regions (where the membrane became detached from the channel wall) prior to forming a single domain at each tip of the confined GUV (Figure top, Movie S1). When the same experiment was performed with a composition DPPC/DOPC/Chol = 40/35/25 mol%, some GUVs also phaseseparated but with a Ld-rich membrane (Figure bottom, Movie S2). Interestingly, despite a different lipid composition, it was again the phase in minority, in this case Lo, that accumulated and grew at the free regions to form a single domain at each GUV extremity.