Members of the genus Orthoflavivirus (family Flaviviridae) are positive-sense RNA viruses that cause a variety of human diseases, including fever, encephalitis, hepatitis, microcephaly, and hemorrhagic fevers. Many of these viruses are insect-borne, including mosquito-borne West Nile virus (WNV) and Zika virus (ZIKV) and tick-borne Powassan virus (POWV). The aim of this study was to investigate molecular determinants of orthoflavivirus infections using different models, such as induced pluripotent stem cell (iPSC)-derived human neural progenitor cells (hNPCs) and cell lines originating from different species and tissues. iPSC-derived hNPCs have been widely used as a model to study how ZIKV infections induce cell cycle arrest and oxidative stress; however, a direct comparison between hNPC infections by WNV, ZIKV, and POWV has not previously been performed. Here, we observed that WNV, ZIKV, and POWV all readily infect hNPCs and induce cell cycle arrest, apoptotic signaling, accumulation of reactive oxygen species (ROS), oxidation of nuclear and mitochondrial DNA, and double-stranded breaks (DSB) in nuclear DNA. While infections by these viruses resulted in similar cellular responses, the effects occurred with different kinetics, with the WNV infections proceeding the fastest and the ZIKV and POWV infections slower. Infections by each of these viruses also induced a cellular antioxidant response, with the POWV infection inducing higher levels of reduced glutathione (GSH) than the WNV or ZIKV infections. In addition, molecular mechanisms of the mouse orthoflavivirus resistance gene Oas1b were investigated. Some mouse strains show resistance to orthoflavivirus infections and disease that is mediated by Oas1b by an unknown mechanism. A mass spectrometry-based proteomics analysis revealed that immunoprecipitation of Oas1b from WNV-infected cell lysates primarily enriched viral proteins and the cellular proteins Alg12 (a critical enzyme involved in N-linked glycosylation) and UFM1 (a ubiquitin-like modifier that covalently binds to proteins as a post-translational modification to target them for ER-phagy processes). Despite significant enrichment of viral proteins in the mass spectrometry-based experiments, neither viral proteins nor cellular proteins Alg12 and UFM1 were detected by western blotting in Oas1b immunoprecipitation experiments, possibly indicating indirect interactions. Finally, Oas1b-mediated orthoflavivirus resistance was not found to depend on N-linked glycosylation of the viral glycoproteins prM and NS1.