The phase separation of aminophospholipids in glycerophospholipid matrix and the effect of cholesterol were studied by means of fluorescence microscopy of giant unilamellar vesicles (GUV). GUVs were composed of binary mixtures, egg yolk phosphatidylcholine (eggPC)/egg yolk phosphatidylethanolamine (eggPE) and egg yolk phosphatidylcholine (eggPC)/brain phosphatidylserine (brainPS), and ternary ones with both aminophospholipids (eggPC/eggPE/brainPS). Gel/liquid-disordered phase coexistence was detected in these mixtures, where aminophospholipids segregate in gel leaf-like domains. When cholesterol (CHOL) was added, the phase separation was shifted at lower temperatures. CHOL increases miscibility of aminophospholipids in PC matrix. Addition of PE and PS to the ternary mixtures (eggPC/eggSM/CHOL) induced liquid-ordered domain formation at higher temperatures. Based on these results, one can conclude that aminophospholipids promote the formation of Lo domains.