The mating-type (mat) locus of Neurospora crassa has a dual function. It is required for mating during sexual development, but it also mediates heterokaryon incompatibility following hyphal fusion during vegetative growth. Previously, it was determined that mutations in vib-1, which encodes a putative transcriptional regulator, suppress het-c heterokaryon incompatibility. This study showed that mutations in vib-1 suppress mat heterokaryon incompatibility in A/a partial diploids; suppression in (A+a) heterokaryons was variable and affected by unknown genetic factors. tol, a gene required for mat heterokaryon incompatibility, is epistatic to vib-1 in mediating mat heterokaryon incompatibility. vib-1 and tol also display a hierarchical order of strength of heterokaryon incompatibility that affected fertilization patterns. In addition to its role in heterokaryon incompatibility, vib-1 also negatively regulates conidiation. This study shows that FL, a positive regulator of conidiation, may partially activate conidiation by repressing VIB-1 function. VIB-1 may also partially regulate heterokaryon incompatibility mediated by genetic differences at het-e and het-8 because mutations in vib-1 increased the survival rate of self-incompatible het-8 and het-e partial diploid progeny.