Introduction: In vitro toxicology evaluations utilizing human stem cell models represent attractive alternatives to conventional animal models, which are not always predictive of human responses. Silver nanoparticles (AgNP) are a potent antimicrobial for use in orthopedic devices. However, AgNP exposure may alter the behavior of stem cells within the bone marrow. The aim of this study was to determine if differences in cell proliferation, early, and late osteogenic differentiation markers can be detected in osteogenically differentiating human bone marrow mesenchymal stem cells (hBMSC) over a 21-day period at AgNP concentrations that are not considered cytotoxic after 24-hour exposure per ISO 10993-5 cytotoxicity testing guidelines. Materials and Methods: Polyvinylpyrrolidone (PVP)-coated 10 nm AgNP were evaluated for their short-term (24-hour) cytotoxicity to hBMSC using the MTT assay to determine subcytotoxic concentrations for the subsequent long-term study investigating osteogenic differentiation. hBMSC were exposed to 1, 5, or 10 lg/mL AgNP in three different exposure scenarios: single (24-hour), repeated (24-hour at 1, 7, and 14 days), or continuous (21day) exposure. Alkaline phosphatase (early osteogenic differentiation marker), hydroxyapatite deposition (late marker), and cell proliferation were measured at days 1, 7, 14, and 21. Results: AgNP exposure reduced cell proliferation for all treatments. Neither differentiation marker expression was observed at any timepoint in hBMSC exposed to AgNP at 10 lg/mL for any exposure scenario or in cells exposed to 5 lg/mL in the repeated or continuous exposure scenarios. Conclusions: Differences in proliferation and osteogenic marker expression were detected in the different exposure scenarios at AgNP concentrations identified as subcytotoxic in a 24-hour exposure assay.