Human cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, but reactivation from latency in immunocompromised subjects results in serious disease. Latency and reactivation are defining characteristics of the herpesviruses and are key to understanding their biology; however, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. Here we present evidence, based entirely on direct analysis of material isolated from healthy virus carriers, to show that myeloid dendritic cell (DC) progenitors are sites of HCMV latency and that their ex vivo differentiation to a mature DC phenotype is linked with reactivation of infectious virus resulting from differentiation-dependent chromatin remodeling of the viral major immediate-early promoter. Thus, myeloid DC progenitors are a site of HCMV latency during natural persistence, and there is a critical linkage between their differentiation to DC and transcriptional reactivation of latent virus, which is likely to play an important role in the pathogenesis of HCMV infection.