How multicellularity with division of labor between cell types evolved is one of the most fundamental questions in biology, but previous studies have hardly explored single-gene pre-adaptations that may allow differentiation to be realized. We show that differential expression of FLO11 produces an evolutionary stable division of labor trait in clonal S. cerevisiae biofilm colonies on medium with intermediate viscosity. Differentiated Flo11+/- colonies obtain a 4-fold growth advantage over undifferentiated colonies by overgrowing glucose resources instead of depleting them as undifferentiated Flo11- colonies do, and they maintain their differentiated state by switching non-adhesive cells to adhesive cells with predictable probability in the growing edge. Our results demonstrate how differentiation allows fitness advantages by preemptive territory acquisition and robustness against invading free-riding strains. S. cerevisiae biofilm thus represents a suitable model for studying the very first evolutionary steps towards clonal multicellularity.