American Hophornbeam (Ostrya virginiana (Mill.) K. Koch) is an underutilized ornamental landscape tree. It has desirable ornamental features including exfoliating bark and unique fruit clusters that resemble the fruit of hops (Humulus lupulus L.). The recent decline in ash (Fraxinus spp.), as a result of the emerald ash borer, has increased American Hophornbeam’s importance as it is a relatively pest-free species. Currently, American Hophornbeam is seed propagated, with no clonal propagation reported for the species. Objective of this research was to study the effect of different basal nutrient media and concentrations of 6-benzylaminopurine (BAP) for axillary shoot proliferation and concentration of IBA for axillary root induction. Four nutrient basal nutrient media (Murashige and Skoog, MS; Long et al., LP; Driver and Kuniyuki, DKW; and Lloyd and McCown, WPM) and five concentrations (0, 2, 4, 8, and 10 μM) of BAP were used to determine proliferation of viable axillary shoots. There was a significant interaction between basal nutrient media and BAP concentration, with DKW medium plus 8 μM BAP producing the most axillary shoots (4.6) and propagules (6.2) per nodal segment as compared to other combinations evaluated. In vitro rooting was evaluated utilizing DKW medium and four concentrations (0, 0.5, 1, and 2 μM) of indole-3-butyric acid (IBA). IBA at 0.5, 1, and 2 μM produced significantly more roots (1.8, 2.4, 2.5, respectively) than the control (0.4). Concentrations of 0.5 and 1 μM IBA produced significantly longer roots (2.35 and 3.04, respectively) than all other evaluated treatments. IBA had a significant impact on the survivability during greenhouse acclimatization. Only axillary shoots that were exposed to IBA survived (100%) as compared to the control (0%).