The extracellular matrix is a dynamic and active component of the mesenchymal stem cell niche, which controls their differentiation and self-renewal. Traditionalin vitroculture systems are not able to mimic matrix-cell interactions due to the small amount of extracellular matrix present. Macromolecular crowding, a biophysical phenomenon based on the excluded-volume effect, dramatically accelerates and increases tissue-specific extracellular matrix deposition duringin vitroculture. Herein, the influence of macromolecular crowding in pre-condition and tri-lineage differentiation of human bone marrow mesenchymal stem cells was investigated. Carrageenan, a sulphated polysaccharide, enhanced chondrogenesis, as evidenced by increased collagen type II and chondroitin sulphate deposition and unaffected Sox-9 expression. Osteogenesis was also enhanced when carrageenan was used only in the differentiation phase, as evidenced by increased mineralisation, collagen type I deposition and osteopontin expression. Adipogenesis was not enhanced in the presence of carrageenan, suggesting that the chemistry of the crowder may affect stem-cell-lineage commitment. In conclusion, carrageenan, a sulphated polysaccharide, enhanced extracellular matrix deposition and promoted chondrogenesis and osteogenesis but not adipogenesis in human bone marrow mesenchymal stem cell cultures.