Biofilms are sessile communities composed of aggregates of cells that are encased in extracellular polymeric matrix of their own synthesis. The formation of biofilms has been proposed to be a developmental process wherein planktonic bacteria adapt to life on a surface. Here, we report that biofilm formation in Pseudomonas aeruginosa occurs in stages and that this bacterium displays multiple phenotypes during development as a biofilm. We determined the onset and abundance of biofilm-stage specific proteins over the course of biofilm development. Onset of protein abundance was found to correlate with the progression of biofilms in developmental stages. Protein identification revealed that proteins with similar function grouped within similar protein abundance patterns. Metabolic proteins were found to group within a pattern separate from virulence, antibiotic resistance, and quorum sensing related proteins. In addition, we determined by 2D/PAGE combined with immunoblot analysis and by LC-MS-MS analysis in conjunction with cICAT labeling that P. aeruginosa displays multiple protein phosphorylation phenotypes during development as a biofilm. Mutations in two genes encoding for phosphorylated regulatory proteins did not confer defects in growth, initial attachment, early biofilm formation, or twitching motility but were observed to arrest biofilm development. Specifically, we have identified a regulatory protein controlling the transition from initial attachment to the cluster formation stage, and one regulatory protein controlling the transition from the cluster formation to the microcolony formation stage. In addition, inactivation of respective regulatory proteins resulted in altered phosphoryaltion patterns over the course of biofilm formation.