Reassortment of their segmented genomes allows influenza A viruses (IAV) to gain new characteristics, which potentially enable them to cross the species barrier and infect new hosts. Improved replication was observed for reassortants of the strictly avian IAV A/FPV/Rostock/34 (FPV, H7N1) containing the NS segment from A/goose/Guangdong/1/1996 (GD, H5N1), but not for reassortants containing the NS segment of A/Mallard/NL/12/2000 (MA, H7N3). The NS1 of GD and MA differ only in 8 amino acid positions. Here, we show that efficient replication of FPV-NSMA-derived mutants was linked to the presence of a single substitution (D74N) and more prominently to a triple substitution (P3S+R41K+D74N) in the NS1MA protein. The substitution(s) led to (i) increased virus titers, (ii) larger plaque sizes, and (iii) increased levels and faster kinetics of viral mRNA and protein accumulation in mammalian cells. Interestingly, the NS1 substitutions did not affect viral growth characteristics in avian cells. Furthermore, we show that a FPV mutant with N74 in the NS1 (already possessing S3+K41) is able to replicate and cause disease in mice, demonstrating a key role of NS1 in the adaptation of avian IAV to mammalian hosts. Our data suggest that (i) adaptation to mammalian hosts does not necessarily compromise replication in the natural (avian) host and (ii) very few genetic changes may pave the way for zoonotic transmission. The study reinforces the need for close surveillance and characterization of circulating avian IAV to identify genetic signatures that indicate a potential risk for efficient transmission of avian strains to mammalian hosts.