The filamentous fungus Fusarium oxysporum causes vascular wilt on a wide range of plant species and is an emerging pathogen of humans. A mitogen-activated protein kinase, Fmk1, and a G protein beta subunit, Fgb1, control pathogenicity of F. oxysporum on plants through distinct signalling pathways. In the present report, we studied the genetic interaction between fmk1 and fgb1 and their role in virulence on a mammalian host. The delta fmk1 or delta fgb1 single mutants exhibited similar virulence patterns as the wild type strain in an immunodepressed mouse model. By contrast, double mutants lacking both genes had dramatically reduced virulence. All mutants showed similar in vitro growth or tolerance to temperature and osmotic stress as the wild type strain. However, the delta fgb1 and delta fmk1 strains were reduced in specific extracellular protease activity or adhesion to fibronectin, respectively, two factors previously associated with fungal virulence. Thus, Fmk1 and Fgb1 are components of distinct signalling pathways which collectively control virulence of F. oxysporum on mammalian hosts.