Anchorage dependent cells can sense and respond to extracellular matrix (ECM) stiffness, but stiffness gradients are often found in vivo via normal tissue variation or pathological conditions, such as the post-infarct myocardial scar which is several folds stiffer than healthy tissue. We have previously shown that mesenchymal stem cell (MSC) differentiation as well as migration is regulated by substrate stiffness in 2D in vitro; it is important to determine if this also occurs in 3D as it could explain MSC migration and calcification in infarct scars in vivo. 3 mg/ml collagen hydrogels were crosslinked with 1, 5, 10, and 20 mM genipin, a natural nontoxic collagen crosslinker, for 2, 4, 12, 24, and 48 hours to yield physiological stiffnesses ranging from 0.9 to 6.4 kPa. MSC behavior on these matrices mimicked previous reports. 3D gradient hydrogels with encapsulated MSCs, fabricated using microfluidics to diffuse genipin through a cell-collagen network, should result in a spatial gradient of crosslinking across the gel. MSCs proliferation, migration, morphology, and differentiation in the presence of this 3D gradient will be compared with 2D gradients in which MSCs were observed to first migrate and then differentiate. MSC durotaxis during wound healing in vivo may be additionally accompanied by haptotaxis, migration due to a matrix ligand gradient, as a result of localized matrix secretion by fibroblasts at the site of regeneration. 2D haptotatic hydrogels with gradients of collagen and fibronectin were also made and can be overlayed onto stiffness gradient hydrogels in order to investigate the effect of corresponding and opposing gradients on MSC behavior. These data show that MSC migration and subsequent differentiation can be regulated by a variety of ECM stimuli in addition to growth factor-mediated pathways.