The complex lifestyle of Pseudomonas aeruginosa, characterized by transitions between free-swimming and surface-associated growth, enables this opportunistic human pathogen to elicit severe acute infections and to establish chronic infections via the development of biofilm communities. The main objective of the present work was to characterize the regulatory events underlying the initiation and progression of biofilm development. We demonstrated that P. aeruginosa cells exhibit distinct protein phosphorylation patterns during free-swimming growth and at various stages of biofilm development, with multiple sensory and regulatory proteins being phosphorylated in a planktonic- or biofilm-specific manner. Phosphorylation timing of at least five such proteins correlated with their functions in the motile-sessile switch and biofilm formation. Inactivation of three novel regulatory proteins, BfiS (PA4197), BfmR (PA4101), and MifR (PA5511), which are sequentially phosphorylated during biofilm formation, arrested biofilm development at stages corresponding to their respective phosphorylation onset. Characterization of the mutant strains further established the stage-specific roles of these regulators: early biofilm regulator BfiS coordinates quorum sensing and post-transcriptional regulatory events; BfmR is involved in early biofilm maturation by controlling cell lysis and DNA release; and MifR enables transition to later maturation stages by modulating adaptation mechanisms for long term biofilm cell survival under anaerobic conditions. In addition to demonstrating stage-specific regulatory events over the course of biofilm development, the present work also revealed that at least three novel planktonic-specific regulators, PlkR (PA0701), PlrS (PA2824), and DpgS (PA2583), are involved in promoting or preserving the free-swimming mode of growth. Furthermore, the work underscored the complexity of sensor/regulator hybrid signaling by demonstrating that the hybrids GacS, PlrS, and DpgS can exert multiple and opposite regulatory effects depending on phosphorylation state and/or stage of biofilm formation. Finally, analyses of the novel planktonic- and biofilm-specific regulatory systems revealed that a combination of aspects including virulence factor production, surface colonization, and dynamics of biofilm development determine the progression of in vivo infections. Taken together, the present findings demonstrated that highly coordinated and sequential signaling events underlie the transitions between planktonic and biofilm modes of growth and regulate the stage-specific mechanisms during the developmental progression of biofilm maturation.