Biofilm formation by the Gram-positive bacterium Bacillus subtilis is tightly controlled at the level of transcription. The biofilm contains specialised cell types that arise from controlled differentiation of the resident isogenic bacteria. DegU is a response regulator that controls several social behaviours exhibited by B. subtilis including swarming motility, biofilm formation and extracellular protease (exoprotease) production. Here, for the first time, we examine the prevalence and origin of exoprotease-producing cells within the biofilm. This was accomplished using single-cell analysis techniques including flow cytometry and fluorescence microscopy. We established that the number of exoprotease- producing cells increases in prevalence as the biofilm matures. This is reflected by both an increase at the level of transcription and an increase in extracellular protease activity over time. We go on to demonstrate that exoprotease- producing cells arise from more than one cell type; namely matrix-producing and non-matrix producing cells. In toto these findings allow us to add exoprotease-producing cells to the list of specialised cell types that are derived during B. subtilis biofilm formation and furthermore the data highlights the plasticity in the origin of differentiated cells.