Biofilm formation by the Gram-negative gammaproteobacteriumPseudomonas fluorescensrelies on the production of the repeat-in-toxin (RTX) adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the production of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by post-transcriptionally regulating the production of LapA and MapA. We find that a ΔfleQmutant has a biofilm formation defect compared to the WT strain, which is attributed in part to a decrease in LapA and MapA production, despite the ΔfleQmutant having increased levels oflapAandmapAtranscripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that over-stimulation of the Gac/Rsm pathway partially rescues biofilm formation in the ΔfleQmutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by post-transcriptionally regulating the production of LapA and MapA, and that activation of the Gac/Rsm pathway can enhance biofilm formation byP. fluorescens.