Cartilage tissue engineering strategies seek to repair damaged tissue using approaches that include scaffolds containing components of the native extracellular matrix (ECM). Articular cartilage consists of glycosaminoglycans (GAGs) which are known to sequester growth factors. In order to more closely mimic the native ECM, this study evaluated the chondrogenic differentiation of mesenchymal stem cells (MSCs), a promising cell source for cartilage regeneration, on fibrous scaffolds that contained the GAG-mimetic cellulose sulfate. The degree of sulfation was evaluated, examining partially sulfated cellulose (pSC) and fully sulfated cellulose (NaCS). Comparisons were made with scaffolds containing native GAGs (chondroitin sulfate A, chondroitin sulfate C and heparin). Transforming growth factor-beta3 (TGF-β3) sequestration, as measured by rate of association, was higher for sulfated cellulose-containing scaffolds as compared to native GAGs. In addition, TGF-β3 sequestration and retention over time was highest for NaCS-containing scaffolds. Sulfated cellulose-containing scaffolds loaded with TGF-β3 showed enhanced chondrogenesis as indicated by a higher Collagen Type II:I ratio over native GAGs. NaCS-containing scaffolds loaded with TGF-β3 had the highest expression of chondrogenic markers and a reduction of hypertrophic markers in dynamic loading conditions, which more closely mimic in vivo conditions. Studies also demonstrated that TGF-β3 mediated its effect through the Smad2/3 signaling pathway where the specificity of TGF-β receptor (TGF- βRI)-phosphorylated SMAD2/3 was verified with a receptor inhibitor. Therefore, studies demonstrate that scaffolds containing cellulose sulfate enhance TGF-β3-induced MSC chondrogenic differentiation and show promise for promoting cartilage tissue regeneration.