It has been suggested that the hemagglutinin of the human-infecting cattle-derived 2.3.4.4b virus A/Texas/34 H5N1 (H5TX) requires only one mutation, namely Q226L, to switch from binding avian-type to human-type receptor preference. In this study, we examined the binding of H5TX Q226L, along with other key mutations, to sections of human trachea. We conclude that, while H5TX Q226L can bind human-type receptors, more than a single mutation is required for this protein to bind to human respiratory tract tissue. We also report changes in receptor-binding specificity of another 2.3.4.4b HA mutant, H5FR Q226L (from A/duck/France/161108/16 H5N8), associated with the presence of a multibasic cleavage site. This study offers insight into the determinants of evolution toward human-type receptor binding in currently circulating H5Nx viruses. It also emphasizes the importance of testing individual strains using additional methods, including tissue-based approaches, alongside synthetic glycans.IMPORTANCECurrently, H5N1 influenza A viruses are responsible for numerous zoonotic spillover events, from infecting birds to other mammals, including dairy cattle. Although no human-to-human transmission has been observed, several people have been infected. This host range expansion is typically linked to changes in one of the viral surface proteins, hemagglutinin, which can switch its preference from avian-type to human-type receptors. To better understand the potential of the currently circulating H5N1 virus to transmit among humans, we evaluated the effects of the Q226L mutation, in combination with other amino acid substitutions, on binding to the human trachea. We also studied the effect of the multibasic cleavage site, a specific motif present in highly pathogenic influenza strains, on receptor-binding properties. These findings provide insight into the role of receptor binding in influenza infections.