Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa) are important human pathogens that can be found co-infecting the same sites. While Pa remains the major cause of chronic life-threatening lung infections in people with cystic fibrosis, Sa is now the most commonly isolated bacteria from respiratory samples of these patients and co-infection is associated with worse patient outcome. Many in vitro studies have shown Pa factors can lyse or kills Sa, while we and others have observed mucoidy, and the concomitant decrease in the expression of some Pa virulence factors, promotes coexistence with Sa. However much less is known about the Sa factors that promote coexistence. To address this, our laboratory performed a survey of 64 CF isolates using a co-culture assay to determine the ability of Sa to coexist with Pa. We found different levels of sensitivity to Pa but our studies did not reveal any particular Sa genotype (sequence, agr, or spa type) or phenotype (mucoidy, antibiotic resistance profile, or hemolytic activity) associated with coexistence. However, since we found that some Sa survived better or worse than others in the presence of Pa, it suggests that there are Sa genetic factors responsible for tolerance vs. sensitivity vs. hypersensitivity. To begin to identify specific genes involved in Sa’s adaptation to Pa, we performed an experimental evolution study with the standard laboratory strains, Sa JE2 and Pa PAO1. We found that Sa populations could adapt to the negative selective pressures presented by Pa under standard laboratory conditions. We identified that mutation in the aspartate transporter, GltT, promoted coexistence suggesting availability of nutrients can be considered a selective pressure driving competition between Sa and Pa. Further characterization of the interactions between these two microbes may identify targets of interest in each of these pathogens.