Genes flbA-E are involved in sporulation and vegetative growth in Aspergillus nidulans. Inactivation of either of these genes results in a fluffy phenotype with delayed or even abolished sporulation. Previously, a non-sporulating phenotype was obtained by inactivating flbA in Aspergillus niger, which was accompanied by lysis, thinner cell walls, and an increased secretome complexity. Here, we further studied the role of the flb genes of A. niger . Strains Δ flbA, Δ flbB and Δ flbE showed increased biomass formation, while inactivation of flbA-D reduced, or even abolished, formation of conidia. Strain Δ flbA was more sensitive to H 2 O 2 , DTT, and the cell wall integrity stress compounds SDS and Congo Red (CR). Also, Δ flbC was more sensitive to SDS, while Δ flbB , Δ flbD , and Δ flbE were more sensitive to CR. On the other hand, inactivation of flbE increased resistance to H 2 O 2 . Enzyme secretion was impacted when the Δ flb strains were grown on xylose. Strain Δ flbE showed reduced xylanase, cellulase and amylase secretion. On the other hand, amylase secretion at the periphery of the Δ flbA colony was reduced but not in its center, while secretion of this enzyme was increased in the center of the Δ flbB colony but not at its periphery. Inactivation of flbC and flbD also impacted zonal cellulase and amylase activity. Together, the Flb protein family of A. niger function in biomass formation, sporulation, stress response, and protein secretion.