The sensitivity of stem cells to environmental perturbations has prompted many studies which aim to characterize the influence of mechanical factors on stem cell morphogenesis and differentiation. Hydrodynamic cultures, often employed for large scale bioprocessing applications, impart complex fluid shear and transport profiles, and influence cell fate as a result of changes in media mixing conditions. However, previous studies of hydrodynamic cultures have been limited in their ability to distinguish confounding factors that may affect differentiation, including modulation of embryoid body size in response to changes in the hydrodynamic environment. In this study, we demonstrate the ability to control and maintain embryoid body (EB) size using a combination of forced aggregation formation and rotary orbital suspension culture, in order to assess the impact of hydrodynamic cultures on ESC differentiation, independent of EB size. Size-controlled EBs maintained at different rotary orbital speeds exhibited similar morphological features and gene expression profiles, consistent with ESC differentiation. The similar differentiation of ESCs across a range of hydrodynamic conditions suggests that controlling EB formation and resultant size may be important for scalable bioprocessing applications, in order to standardize EB morphogenesis. However, perturbations in the hydrodynamic environment also led to subtle changes in differentiation toward certain lineages, including temporal modulation of gene expression, as well changes in the relative efficiencies of differentiated phenotypes, thereby highlighting important tissue engineering principles that should be considered for implementation in bioreactor design, as well as for directed ESC differentiation.