This rapid communication describes the generation and characterization of a recombinant influenza A/PR/8/34 virus (delNS1) lacking the NS1 gene, which encodes the NS1 protein. The NS1 protein is known to play a crucial role in inhibiting interferon (IFN)-mediated antiviral responses in host cells. The delNS1 virus was generated using reverse genetics and was found to be viable and able to replicate in Vero cells but showed reduced replication in MDCK cells and embryonated chicken eggs. The virus was also nonpathogenic in wild-type mice but caused severe disease in STAT1−/− mice, which lack IFN signaling. Additionally, the delNS1 virus was shown to stimulate transcription from an IFN-regulated promoter, indicating its impaired ability to inhibit the IFN response. These findings suggest that the NS1 protein of influenza A virus is an auxiliary (virulence) factor that plays a key role in inhibiting IFN-mediated antiviral responses. The study also highlights the potential of using "gene knockout" viruses for vaccine development and the therapeutic induction of antiviral states.This rapid communication describes the generation and characterization of a recombinant influenza A/PR/8/34 virus (delNS1) lacking the NS1 gene, which encodes the NS1 protein. The NS1 protein is known to play a crucial role in inhibiting interferon (IFN)-mediated antiviral responses in host cells. The delNS1 virus was generated using reverse genetics and was found to be viable and able to replicate in Vero cells but showed reduced replication in MDCK cells and embryonated chicken eggs. The virus was also nonpathogenic in wild-type mice but caused severe disease in STAT1−/− mice, which lack IFN signaling. Additionally, the delNS1 virus was shown to stimulate transcription from an IFN-regulated promoter, indicating its impaired ability to inhibit the IFN response. These findings suggest that the NS1 protein of influenza A virus is an auxiliary (virulence) factor that plays a key role in inhibiting IFN-mediated antiviral responses. The study also highlights the potential of using "gene knockout" viruses for vaccine development and the therapeutic induction of antiviral states.