Multidrug-resistantPseudomonas aeruginosa(MDRP) is one of the most important pathogens in clinical practice. To clarify the mechanisms contributing to its emergence, we isolated MDRPs using theP. aeruginosaPAO1, the whole genome sequence of which has already been elucidated. Mutant strains resistant to carbapenems, aminoglycosides, and new quinolones, which are used to treatP. aeruginosainfections, were isolated; however, none met the criteria for MDRPs. Then, PAO1 strains were exposed to these antimicrobial agents in various orders and the appearance rate of MDRP varied depending on the order of exposure; MDRPs more frequently appeared when gentamicin was applied before ciprofloxacin, but were rarely isolated when ciprofloxacin was applied first. Exposure to ciprofloxacin followed by gentamicin increased the expression of MexCD-OprJ, an RND-type multidrug efflux pump, due to the NfxB mutation. In contrast, exposure to gentamicin followed by ciprofloxacin resulted in more mutations in DNA gyrase. These results suggest that the type of quinolone resistance mechanism is related to the frequency of MDRP and that the risk of MDRP incidence is highly dependent on the order of exposure to gentamicin and ciprofloxacin.