Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley. In a previous study, we identified several mutants with reduced virulence by insertional mutagenesis. A transducin beta-like gene named FTL1 was disrupted in one of these nonpathogenic mutants. FTL1 is homologous to Saccharomyces cerevisiae SIF2, which is a component of the Set3 complex involved in late stages of ascospore formation. The Δftl1 mutant was significantly reduced in conidiation and failed to cause typical disease symptoms. It failed to colonize the vascular tissues of rachis or cause necrosis on the rachis of inoculated wheat heads. The Δftl1 mutant also was defective in spreading from infected anthers to ovaries and more sensitive than the wild type to plant defensins MsDefl and osmotin. However, the activation of two mitogen-activated protein kinases, Mgvl and Gpmk1, production of deoxynivalenol, and expression of genes known to be important for plant infection in F. graminearum were not affected, indicating that the defect of the Δftl1 mutant in plant infection is unrelated to known virulence factors in this pathogen and may involve novel mechanisms. The Δftl1 deletion mutant was significantly reduced in histone deacetylation, and many members of the yeast SeO complex are conserved in F. graminearum. FTLl appears to be a component of this well-conserved protein complex that plays a critical role in the penetration and colonization of wheat tissues.