In our previous work, we have produced H5pp using the H5-HA of A/Cambodia/40808/2005 (H5Cam), which was isolated from a patient with a lethal infection of H5N1 virus [18] . In the current study, we have analyzed the ability of H5-HAs from different clades of avian influenza virus to pseudotype lentiviral particles and have found that they do not give rise to the same level of efficient H5pp production when compared with H5Cam. In particular, we have carried out a detailed comparison of the expression and cleavage of two H5-HAs, i.e., H5-HA of A/Anhui/ 1/2005 (H5Anh) and H5Cam, and of their ability to pseudotype lentiviral vector in HEK293T cells. Through several independent lines of evidence we have identified the molecular determinants in H5-HA for efficient incorporation into H5pp envelope and have delineated the underlying mechanism. Our results are discussed in the context of the understanding of human host adaptation of avian influenza H5N1 viruses.