As cardiac-fated cells traverse the course of differentiation into cardiac myocytes, the sequence, magnitude, and spatiotemporal map of biomechanical signals that may influence cardiogenesis have not been fully explored. Whereas several studies have examined the induction of cardiogenesis on different extracellular matrix proteins and substrates, none have examined the effects of substrate stiffness prior to the onset of cardiac differentiation in cardiac-fated cells. We investigate the effects of substrate stiffness on precardiac cell behaviors in an in vitro setting. The cells in the anterior portion of the primitive streak are fated to form the heart, and we show differing levels of smooth muscle ?-actin expression (and E-cadherin) on substrates of differing moduli, which suggests that substrate stiffness may play a role in cardiac differentiation. However, we could not detect changes in the presence of sarcomeric myosin heavy chain expression or the occurrence of beating cultures, which suggests that although cardiac-fated cells show a sensitivity to substrate stiffness, different or more sensitive tests would be required to confirm a functional difference at this early stage.