The bone marrow harbors multipotent mesenchymal stromal cells (MSCs) that nurture hematopoietic stem cells (HSCs). The extracellular matrix (ECM) is an integral part of the bone marrow, and the aim of this study was therefore to examine the effect of engineered ECM substrates on MSC gene expression over time and to determine quantitatively the functional ability of ECM-cultured MSCs to support HSCs. ECMs were surface immobilized using thin films of maleic anhydride to covalently immobilize tropocollagen or fibrillar collagen type I to the substrate. Where indicated, collagen type I fibrils were supplemented with heparin or hyaluronic acid. All surfaces maintained MSC viability and supported cell expansion. Microarray analysis of MSCs cultured on engineered ECM substrates revealed that culture time, as well as substrate composition, significantly affected expression levels. Based on these studies, it was possible to predict the effect of these substrates on in vitro HSC clonogenicity and self-renewal. The ability to regulate the expression of stromal factors using engineered ECM is exciting and warrants further studies to identify the ECM components and combinations that maximize the expansion of clonogenic HSCs.