This article reviews the development of two attenuated West Nile virus (WNV) variants, WNI-25 and WNI-25A. These variants have lost the neuroinvasion trait of the parental virus. Attenuation was achieved through serial passages in mosquito cells and neutralization escape from WNV-specific monoclonal antibody. Genetic analysis reveals amino acid changes between the parental and each of the variants. The attenuated variants preserve the ability to replicate in mice and geese and to induce a protective immune response. WNI-25A was found to be a genetically stable virus. This variant was successfully used as a live vaccine to protect geese against a wild-type virulent WNV field isolate that closely resembles the WNV isolated during the 1999 New York epidemic.