Several upstream activators required for proper activation of brlA are involved in the development, vegetative growth, toxin production, and pathogenesis of Aspergillus fumigatus. In this study, we characterized the roles of two upstream developmental regulators, A. fumigatus flbB (AfuflbB) and flbE (AfuflbE), in toxin production and virulence. The deletion of AfuflbB and AfuflbE resulted in reduction of the expression of AfulaeA. Moreover, only about 8% to 10% of fumagillin was produced in the two mutants compared with that of wild type, and ΔAfuflbB strain produced 85% of gliotoxin compared with wild type, whereas none was produced by ΔAfuflbE. Flow-cytometric analysis revealed decreased necrotic and apoptotic polymorphonuclear leukocytes cell death after exposure to supernatants from ΔAfuflbB and ΔAfuflbE strains compared with the wild type. These results indicate that FlbB and FlbE are necessary for the proper laeA expression, toxin production, and virulence of A. fumigatus.