Cytomegalovirus (CMV) is a major cause of congenital brain disease, and its neuropathogenesis may be related to viral infection of rapidly dividing, susceptible neural precursor cells (NPCs). In the present study, we evaluated the susceptibility of human fetal brain-derived NPCs (nestin(+), A2B5(+), CD133(+)) to infection with CMV. Data derived from these studies demonstrated that undifferentiated NPCs supported productive viral replication. After differentiation in the presence of serum, a treatment that promotes development of an astroglial cell phenotype (GFAP(+), nestin(-), A2B5(-)), viral expression was retained. However, differentiation of NPCs in medium containing platelet-derived growth factor and brain-derived neurotropic factor, conditions that support the development of neurons (Tuj-1(+), nestin(-), A2B5(-)), resulted in reduced viral expression, with corresponding decreased CMV major immediate-early promoter (MIEP) activity relative to undifferentiated cells. Further experiments showed that cellular differentiation into a neuronal phenotype was associated with elevated levels of various CCAAT/enhancer binding protein beta (C/EBP)-beta isoforms, which suppressed MIEP activity in cotransfected NPCs. Taken together, these data demonstrate that the susceptibility of primary human NPCs to CMV is retained concomitantly with differentiation into glial cells but is actively repressed following differentiation into neurons.