Fungal sporulation is separated from its vegetative growth. In this paper a novel two-stage cultivation method is described to determine fungal nutrition requirements for maximizing spore production. In this method, the fungus is first cultured on agar to grow vegetatively for 4d, and then transferred to a new nutritionally defined medium, where it sporulates for 4d. The effects of media nutrients on spore production were compared using the new method vs. traditional continuous cultivation for six fungi with biological control potential Paecilomyces lilacinus, Pochonia chlamydosporia, Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii, and Trichoderma viride. The two methods did not differ for P. lilacinus or M. anisopliae but differ for the other fungi. In particular, B. bassiana and P. chlamydosporia required different nutrients to produce large numbers of spores with continuous vs. two-stage cultivation. Spore production by L. lecanii differed greatly between the two cultivation methods when sucrose and yeast extract were as carbon and nitrogen sources, respectively. It was revealed yeast extract supported abundant sporulation of T. viride by two-stage cultivation. Based on the new method, T. viride produced the most spores when the medium contained 2g carbon/L, C:N=10:1, and cellobiose/yeast as the carbon/nitrogen sources.