The factors selected to optimize the productivity of Pleurotus flabellatus biomass in 250ml working volume Erlenmeyer flask were agitation rate, initial pH value and incubation temperature. The central composite design was applied to study the significant factors and the interactions between the chosen factors, if present. The Design Expert software generated 20 runs. The optimized conditions obtained were as follows: the agitation rate of 129.8 rpm, incubation temperature at 27.8 degree C, and initial pH of 6.06. The optimized conditions tripled the productivity at the range of 980 - 1040 mg/litre/day compared to the initial rate productivity at 310 mg/litre/day. From the quadratic equation,the agitation rate, temperature and the interaction between agitation rate and temperature were found to be significant (p < 0.05). At optimum conditions, the experimental data supported the theoretical estimate.