The Flaviviridae include almost 70 viruses, nearly half of which have been associated with human disease. These viruses are among the most important arthropod-borne viruses worldwide and include dengue, yellow fever, and Japanese encephalitis viruses. Morbidity and mortality caused by these viruses vary, but collectively they account for millions of encephalitis, hemorrhagic fever, arthralgia, rash, and fever cases per year. Most of the members of this family are transmitted between vertebrate hosts by arthropod vectors, most commonly mosquitoes or ticks. Transmission cycles can be simple or complex depending on the hosts, vectors, the virus, and the environmental factors affecting both hosts and viruses. Replication of virus in invertebrate hosts does not seem to result in any significant pathology, which suggests a close evolutionary relationship between virus and vector. Another example of this relationship is the ability of these viruses to grow in invertebrate cell culture, where replication usually results in a steady state, persistent infection, often without cytopathic effect. Yields of virus from insect cell culture vary but are generally similar to yields in vertebrate cells. Replication kinetics are comparable between insect and vertebrate cell lines, despite differences in incubation temperature. Both vertebrate and insect cell culture systems continue to play a significant role in flavivirus isolation and the diagnosis of disease caused by these agents. Additionally, these culture systems permit the study of flavivirus attachment, penetration, replication, and release from cells and have been instrumental in the production and characterization of live-attenuated vaccines. Both vertebrate and insect cell culture systems will continue to play a significant role in basic and applied flavivirus research in the future.