Influenza infections are considered a global threat to public health and cause seasonal epidemics and recurring pandemics. High mutation rates facilitate the generation of viral escape mutants rendering vaccines and drugs directed against virus-encoded targets ineffective. One alternative approach that could prevent viral escape is the targeting of host cell determinants that are temporarily dispensable for the host but crucial for virus replication. Here, we report a genome-wide RNAi screening approach in mammalian cell culture system that led us to the identification of several host cell genes influencing influenza A virus replication. Interestingly, the majority of the identified host gene products are indispensable for viral replication of a broad range of influenza viruses ranging from the highly pathogenic avian H5N1 strain to the current pandemic swine-origin H1N1 strain. Our results provide a new approach to explore virus–host interactions and to identify promising antiviral targets.