A recently isolated thermotolerant microalga Desmodesmus sp. F2 has the traits of becoming potential biodiesel feedstock, such as high growth rate, high lipid content, and quick precipitation. Its overall lipid productivity was 113 mg/L/d when grown under non-optimal conditions using batch cultivation. A two-step response surface methodology was adopted to optimize its cultivation conditions. The overall lipid productivity was increased to 263 mg/L/d when the cells were grown under the optimized conditions of 6.6mM initial nitrogen level and 6 days nitrogen depletion treatment in 700 μmol/m(2)/s light intensity at 35°C using batch cultivation. Fed-batch and semi-continuous cultivations were employed to further increase its lipid productivity to 213 and 302 mg/L/d, respectively. The 302 mg/L/d is the highest overall lipid productivity of microalgae ever reported in the literature. This study provides the information required for the design and operation of photobioreactors for large scale outdoor cultivation of this species.