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Bacteria often live in the form of surface-associated communities of cells termed biofilms. Within biofilms, there is a division of labor in which genetically identical cells differentiate to serve distinct functions. This cellular differentiation results from a response to extracellular signals that occur due to changes in the local environment of a cell or in response to signaling molecules that the cells themselves produce. In this review, we discuss differentiation in biofilms, focusing on the molecular mechanisms that regulate differentiation in the bacterium Bacillus subtilis. In this organism, there is a subpopulation of cells within a biofilm that produces a signal, while a different subpopulation of cells responds to it. Studying what signals cells use to communicate with each other within a biofilm will allow for better design of strategies to prevent and disrupt biofilms.