Velvet domain proteins coordinate developmental processes and secondary metabolism in fungi. In this study, we characterized the role of all four velvet proteins in the life cycle of the plant pathogenic fungus Verticillium dahliae. From other fungi, the formation of different homo-and heterodimers as well as complexes with non-velvet proteins is known. We could show that V. dahliae Vel1 is important for initial plant root colonization. In planta, Vel1 together with Vel3 is involved in fungal distribution by conidia. Disease symptom induction in tomato plants also depends on Vel1. In the senescent plant formation of microsclerotia as resting structure ex planta is controlled by Vel1, Vel2 and Vel3. In vitro protein pull-downs were conducted to identify interaction partners of velvet proteins in V. dahliae. In this study, we were able to identify interactions between Vel1 and Vel2, Vel2 and Vos1, and Vel3 and Vos1 during vegetative hyphae growth, respectively. Similar interactions are known for A. nidulans. Further interacting candidates were identified for the velvet proteins except for Vel3, for which only interaction with Vos1 was found during the tested conditions. Velvet-interacting candidate proteins included redox-regulating proteins and metabolic enzymes, but also proteins involved in energy metabolism and transport.