Saccharomyces cerevisiae generates complex biofilms called mats on low-density (0.3%) agar plates. The mats can be morphologically divided into two regions: (i) hub, the interior region characterized by the presence of wrinkles and channels, and (ii) rim, the smooth periphery. Formation of mats depends on the adhesin Flo11p, which is also required for invasive growth, a phenotype in which the S. cerevisiae yeasts grow as chains of cells that dig into standard-density (2%) agar plates. Although both invasive growth and mat formation depend on Flo11p, mutations that perturb the multivesicular body (MVB) protein sorting pathway inhibit mat formation in a FLO11-independent manner. These mutants, represented by vps27Δ, disrupt mat formation but do not affect invasive growth, FLO11 gene or protein expression, or Flo11p localization. In contrast, an overlapping subset of MVB mutants (represented by ESCRT [endosomal sorting complex required for transport] complex genes such as VPS25) interrupt the Rim101p signal transduction cascade, which is required for FLO11 expression, and thus block both invasive growth and mat formation. In addition, this report shows that mature Flo11p is covalently associated with the cell wall and shed into the extracellular matrix of the growing mat.