The sequence of applied biochemical stimulation in developing ligament tissue cultures in vitro offers the potential to intricately control cell behavior following the template of native tissue development. Previous studies have identified and enhanced ligament tissue development as defined by matrix in-growth, upregulation of mRNA transcripts for metalloproteinase-2 (MMP-2), collagen types I and III, and collagen type I production. We hypothesize that sequential application of growth factors through extended culture will reinforce the effectiveness of basic fibroblast growth factor and transforming growth factor beta (bFGF/TGF-beta) as the optimal growth factor regimen. Bone marrow stromal cells (BMSCs) were seeded on RGD-coupled silk fiber matrices and cultured in bFGF, epidermal growth factor (EGF), or growth factor-free control for the first 5 days of culture. On day 5, cultures were stimulated with TGF-beta supplemented medium for a total of 28 days. Results indicated enhanced matrix in-growth and collagen type I produced with extended culture, most notably in mitogen / TGF-beta-stimulated cultures. Matrix development attained through extended static culture will support future study leading to the transition and addition of mechanical stimulation for optimized ligament tissue production.