It has been suggested that the hemagglutinin of the human-infecting cattle-derived 2.3.4.4b virus A/Texas/34 (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, associated with the presence of a multibasic cleavage site. This study offers insight into the determinants of evolution towards 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.