Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcεRIα+ cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34+ cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2–3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in (∼ 34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose-and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection.