Stromagenesis is a host reaction of connective tissue that, when induced in cancer, produces a progressive and permissive mesenchymal microenvironment, thereby supporting tumor progression. The stromal microenvironment is complex and comprises several cell types, including fibroblasts, the primary producers of the noncellular scaffolds known as extracellular matrices. The events that support tumor progression during stromagenesis are for the most part unknown due to the lack of suitable, physiologically relevant, experimental model systems. In this report, we introduce a novel in vivo-like three-dimensional system derived from tumor-associated fibroblasts at diverse stages of tumor development that mimic the stromagenic features of fibroblasts and their matrices observed in vivo. Harvested primary stromal fibroblasts, obtained from different stages of tumor development, did not retain in vivo stromagenic characteristics when cultured on traditional two-dimensional substrates. However, they were capable of effectively maintaining the tumor-associated stromal characteristics within three-dimensional cultures. In this study, we demonstrate that in vivo-like three-dimensional matrices appear to have the necessary topographical and molecular information sufficient to induce desmoplastic stroma differentiation of normal fibroblasts.