Alteration of lipid raft organization manifesting as phase separation is important for cellular processes, such as signaling and trafficking. Such behaviors and dynamics of lipid membranes can be affected by external stimuli including both physical and chemical stimuli. In this study, we focused on osmotic-tension-induced phase separation. The effects of osmotic tension on the phase behaviors of vesicles consisting of dioleoylphosphocholine (DOPC)/dipalmitoylphosphocholine (DPPC)/cholesterol (Chol) were quantitatively studied at different temperatures by fluorescence microscopy. We determined the ternary phase diagrams and found that tension leads to a shift in the miscibility temperature. Cholesterol plays a key role in determining the extent of this shift. In addition, we found that osmotic tension can enhance the line tension. The physicochemical mechanism of osmotic-pressure-induced phase separation is discussed.