Primary varicella-zoster virus (VZV) infection in humans produces varicella (chickenpox), after which the virus becomes latent in ganglionic neurons. Analysis of the physical state of viral nucleic acid and virus gene expression during latency requires postmortem acquisition of fresh human ganglia. To provide an additional way to study the VZV-host relationship in neurons, we developed an in vitro model of infected differentiated human neural stem cells (NSCs). NSCs were induced to differentiate in culture dishes coated with poly-l-lysine and mouse laminin in the presence of fibroblast growth factor 2 (FGF-2), nerve growth factor (NGF), brain-derived neurotropic factor (BDNF), dibutyryl cyclic AMP, and retinoic acid. Immunostaining with neuronal (MAP2a and β-tubulin), astrocyte (GFAP), and oligodendrocyte (CNPase) markers revealed that differentiated neurons constituted approximately 90% of the cell population. These neurons were maintained in culture for up to 8 weeks. No cytopathic effect (CPE) developed in neurons infected with cell-free VZV (Zostavax vaccine) compared to human fetal lung fibroblasts infected with VZV. Weeks later, VZV DNA virus-specific transcripts (open reading frames [ORFs] 21, 29, 62, and 63) were detected in infected neurons, and dual immunofluorescence staining revealed the presence of VZV IE63 and gE exclusively in healthy-appearing neurons, but not in astrocytes. Neither the tissue culture medium nor a homogenate prepared from VZV-infected neurons produced a CPE in fibroblasts. VZV induced apoptosis in fibroblasts, as shown by activation of caspase 3 and by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, but not in neurons. This model provides a unique in vitro system to study the VZV-neuronal relationship.