Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin, laminin-111, and laminin-521 enabled hPSCs to attach and expand; however, fibronectin ECM either endogenously produced or exogenously added promoted, while laminins inhibited, cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF. Inducible shRNA knockdown of fibronectin prevented Brachyury + mesoderm formation and subsequent hPSC-CM differentiation. Antibodies blocking fibronectin binding to integrin β1, but not α5, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase blocked cardiac differentiation. These results identify fibronectin, laminin-111 and laminin-521 as defined substrates enabling cardiac differentiation of hPSCs and uncover the essential role of fibronectin and downstream signaling pathways in the early stage of hPSC-CM differentiation.