Fusarium graminearum, the primary cause of Fusarium Head Blight (FHB) of wheat and barley in North America, is a homothallic ascomycete capable of outcrossing. Ascospores produced in early spring from perithecia on infested crop debris serve as primary inoculum for the establishment of epidemics. The sexual behavior of this fungus is regulated by the mating type locus MAT1, which is comprised of two linked idiomorphs, (MAT1-1-1 and MAT1-2-1). It is assumed that the products of these two genes interact to form heterodimers that regulate homothallic sexual development. We generated several independent knockout mutants (KOs) of MAT1, MAT1-1-1, and MAT1-2-1 in the PH-1 strain of F. graminearum. As expected, all of the KOs lost their ability to produce ascospores. The MAT1-1-1 KOs, when paired with the MAT1-2-1 KOs, formed fertile perithecia. Markers segregated 1:1 among the progeny of these perithecia, confirming that the MAT1-1-1 and MAT1-2-1 KOs are heterothallic. The MAT1 KOs were unaffected in aggressiveness to point-inoculated winter wheat (susceptible variety Pioneer 2555) in the greenhouse. In contrast, KOs of the individual specificity genes, especially of MAT1-1-1, resulted in significant reductions in aggressiveness and, in most cases, in DON production. These results suggest that the F. graminearum MAT1-1-1 and MAT1-2-1 specificity proteins individually regulate genes that negatively impact the aggressiveness and toxigenicity of the heterothallic strains.