The pathogenicity of avian influenza virus (AIV) is governed primarily by the hemagglutinin (HA) glycoprotein. Highly pathogenic avian influenza viruses contain a highly cleavable HA molecule susceptible to ubiquitous host proteases, such as furin, resulting in severe systemic infections. In contrast, low-pathogenic avian influenza viruses have HAs that require activation by trypsin-like proteases, restricted to specific organs, causing localized and mild infections. We generated highly pathogenic virus variants by serially passaging low-pathogenic waterfowl isolates that originally replicated poorly in domestic poultry. The increased pathogenicity was correlated with a shift from a low-pathogenic to a highly pathogenic motif in the HA cleavage site. These findings suggest that benign viruses maintained in wild waterfowl in nature have the potential to become highly pathogenic variants during circulation and adaptation in chickens. Moreover, the host specificity of AIV is also primarily determined by the HA glycoprotein. AIVs usually replicate less efficiently in humans, whereas human influenza viruses replicate poorly in birds. This host restriction largely reflects differences in receptor-binding specificity of the HA protein. Most AIVs preferentially bind to sialic acid receptors with α2,3-linked galactose (SAα2,3Gal), while human viruses prefer the SAα2,6Gal linkage. Interestingly, both receptor types are expressed in the respiratory epithelium of pigs. Swine serve as "mixing vessels" that facilitate the reassortment of viruses between avian and human strains. These results provide a molecular basis for the key mechanisms underlying the emergence of novel influenza viruses with pandemic potential in humans.