Author summaryVerticillium wilt is a destructive disease of many economic important crop plants induced by fungal plant pathogenic Verticillium spp. such as V. dahliae. Developmental processes and the production of secondary metabolites are coordinated by fungal velvet domain proteins. The four V. dahliae velvet domain proteins form the heterodimers Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 during vegetative growth. Vel1, Vel2 and Vel3 coordinate the formation of the microsclerotia resting structures and the production of the metabolite melanin, which is deposited in microsclerotia. The survival of these resting structures in the soil is essential for Verticillium spp. during the monocyclic disease. Microsclerotia germinate in the presence of a suitable host, the resulting hyphae grow to and colonize the root. We discovered that this important process of initial colonization requires the presence of Vel1. Once inside the plant, the fungus distributes in the vascular system by conidia formation, which depends on Vel1 and Vel3. Vel1 is a promising target to control the fungal disease, because it is required for the induction of disease symptoms.The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-kappa B transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae. Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V. dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V. dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta.