Triacylglycerols of oleaginous algae are promising for production of food oils and biodiesel fuel. Air-drying of cells induces triacylglycerol accumulation in a freshwater green alga, Chlorella kessleri, therefore, it seems that dehydration, i.e., intracellular hyperosmosis, and/or nutrient-limitation are key stressors. We explored this possibility in liquid-culturing C. kessleri cells. Strong hyperosmosis with 0.9 M sorbitol or 0.45 M NaCl for two days caused cells to increase the triacylglycerol content in total lipids from 1.5 to 48.5 and 75.3 mol%, respectively, on a fatty acid basis, whereas nutrient-limitation caused its accumulation to 41.4 mol%. Even weak hyperosmosis with 0.3 M sorbitol or 0.15 M NaCl, when nutrient-limitation was simultaneously imposed, induced triacylglycerol accumulation to 61.9 and 65.7 mol%, respectively. Furthermore, culturing in three-fold diluted seawater, the chemical composition of which resembled that of the medium for the combinatory stress, enabled the cells to accumulate triacylglycerol up to 24.7 weight% of dry cells in only three days. Consequently, it was found that hyperosmosis is a novel stressor for triacylglycerol accumulation, and that weak hyperosmosis, together with nutrient-limitation, exerts a strong stimulating effect on triacylglycerol accumulation. A similar combinatory stress would contribute to the triacylglycerol accumulation in air-dried C. kessleri cells.