Miscibility phase diagrams at different temperatures for (A) DiPhyPC-DPPG-Chol vesicles prepared in water, (B) DiPhyPG-DPPC-Chol vesicles prepared in water, and (C) DiPhyPG-DPPC-Chol vesicles prepared in 10 mM KCl, 5 mM TRIS, and 0.5 mM EDTA. Regions shaded in gray are inaccessible by our method of gentle hydration and fluorescence microscopy. Star symbols represent phase separation that is coexistence of Lo/Ld, solid/Ld, or solid/Lo/Ld phases. This ambiguity arises because Lo and solid phases can be challenging to distinguish by fluorescence microscopy of vesicles, especially when three phases (Ld, Lo, and solid) coexist in the same vesicle, and/or when a temperature quench from the Ld–Lo coexistence region nucleates solid domains.