In this study we present a novel method for studying cellular traction force generation and mechanotransduction in the context of cardiac development. Rat hearts from three distinct stage of development (fetal, neonatal and adult) were isolated, decellularized and characterized via mechanical testing and protein compositional analysis. Stiffness increased similar to 2-fold between fetal and neonatal time points but not between neonatal and adult. Composition of structural extracellular matrix (ECM) proteins was significantly different between all three developmental ages. ECM that was solubilized via pepsin digestion was cross-linked into polyacrylamide gels of varying stiffness and traction force microscopy was used to assess the ability of mesenchymal stem cells (MSCs) to generate traction stress against the substrates. The response to increasing stiffness was significantly different depending on the developmental age of the ECM. An investigation into early cardiac differentiation of MSCs demonstrated a dependence of the level of expression of early cardiac transcription factors on the composition of the complex ECM. In summary, this study found that complex ECM composition plays an important role in modulating a cell's ability to generate traction stress against a substrate, which is a significant component of mechanotransductive signaling. (C) 2013 Elsevier Inc. All rights reserved.