Although a polybasic HA₀ cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA₀ cleavage site have been identified that are low pathogenic in chickens. In this study we generated a reassortant H5N3 virus that possessed the HA gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA₀ cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3 inoculated birds replicated and shed virus and seroconverted, transmission to na?ve contacts did not occur. To determine if this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole genome sequencing demonstrated the fixation of twelve non-synonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low pathogenic rH5N3 increased its virulence, transmission to na?ve contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype.