For many RNA viruses, immunity is triggered when RIG-I-like receptors (RLRs) detect viral RNA. However, only a minority of infected cells undergo innate immune activation. By examining these "first-responder" cells during West Nile virus infection, we found that specific accumulation of antigenomic negative-sense viral RNA (-vRNA) underlies innate immune activation and that RIG-I preferentially interacts with -vRNA. However, flaviviruses sequester -vRNA into membrane-bound replication compartments away from cytosolic sensors. We found that single-stranded -vRNA accumulates outside of replication compartments in first-responder cells, rendering it accessible to RLRs. Exposure of this -vRNA occurs at late time points of infection, is linked to viral assembly, and depends on the expression of viral structural proteins. These findings reveal that, although most infected cells replicate high levels of vRNA, release of -vRNA from replication compartments during assembly occurs at low frequency and is critical for initiation of innate immunity during flavivirus infection.