is a Gram-negative opportunistic pathogen that can infect the lungs of people with cystic fibrosis (CF). The highly viscous mucus in the CF lung, expectorated as sputum, serves as the primary nutrient source for microbes colonizing this site and induces virulence-associated phenotypes and gene expression in several CF pathogens. Here, we characterized the transcriptional responses of three strains during exposure to synthetic CF sputum medium (SCFM2) to gain insight into how this organism interacts with the host in the CF lung. These efforts led to the identification of 881 transcripts differentially expressed by all three strains, many of which reflect the metabolic pathways used by in sputum, as well as altered stress responses. The latter correlated with increased resistance to peroxide exposure after pregrowth in SCFM2 for two of the strains. We also compared the SCFM2 transcriptomes of two CF isolates to that of the acute infection strain, K279a, allowing us to identify CF isolate-specific signatures in differential gene expression. The expression of genes from the accessory genomes was also differentially altered in response to SCFM2. Finally, a number of biofilm-associated genes were differentially induced in SCFM2, particularly in K279a, which corresponded to increased aggregation and biofilm formation in this strain relative to both CF strains. Collectively, this work details the response of to an environment that mimics important aspects of the CF lung, identifying potential survival strategies and metabolic pathways used by during infections. is an important infecting bacterium in the airways of people with cystic fibrosis (CF). However, compared to the other CF pathogens, has been relatively understudied. The significance of our research is to provide insight into the global transcriptomic changes of in response to a medium that was designed to mimic important aspects of the CF lung. This study elucidates the overall metabolic changes that occur when encounters the CF lung and generates a road map of candidate genes to test using and models of CF.