Mechanical properties of the extracellular microenvironment regulate cell behaviors including migration, proliferation, and morphogenesis. Although elastic moduli of synthetic materials have been studied, little is known about the properties of naturally produced extracellular matrix. Here, we utilized atomic force microscopy to characterize the microelastic properties of decellularized cell-derived matrix from human pulmonary fibroblasts. This heterogeneous three-dimensional matrix had an average thickness of 5±0.4 μm and a Young’s modulus of 105±14 Pa. Ascorbate treatment of the lung fibroblasts prior to extraction produced a two-fold increase in collagen I content, but did not affect the stiffness of the matrices compared to matrices produced in standard medium. However, fibroblast-derived matrices that were crosslinked with glutaraldehyde demonstrated a 67% increase in stiffness. This work provides a microscale characterization of fibroblast-derived matrix mechanical properties. An accurate understanding of native three-dimensional extracellular microenvironments will be essential for controlling cell responses in tissue engineering applications.