Each tissue has a certain composition of the ECM. Bone is composed of organic (40%) and inorganic compounds (60%), in which 90% of the organic component is collagen and has a low content of glycosaminoglycan (GAG). Cartilage is composed of 60% collagen and 25‐35% PGs, and IVD is composed of 20‐30 % collagen and 36‐70% PGs, respectively. The appropriate microenvironment also supports and regulates MSCs differentiation towards the specific cell linage.As the major components of ECM, collagen, HA and GAG are important candidates to mimic the native microenvironment. We aimed to fabricate scaffolds that mimic the complex ECM of different tissues and we hypothesized that the biomimetic scaffolds will benefit hMSCs differentiated into specific lineages. Herein, we demonstrate that scaffolds mimicking the ECM composition particularly with different GAG content in terms of GAG/HYP ratios, will facilitate multiple differentiation of hMSCs towards specific lineages. In osteogenic differentiation, the Col (GAG/HYP 0) scaffold showed higher calcium (Ca) and phosphorus (P) deposition and Ca/P ratio, biomimetic ultrastructure, and osteogenic phenotypic expression. In chondrogenic differentiation, aCol‐GAG (GAG/HYP 4.9:1) showed hydration, higher GAG deposition, biomimetic ultrastructure, and higher chondrogenic phenotype expression. In discogenic differentiation, the aCol‐aHA‐GAG (GAG/HYP 19.8:1) showed intensive GAG deposition, biomimetic ultrastructure, and higher phenotypic expression of NPCs. his work demonstrated that the fabrication of scaffold mimicking the natural composition of native tissue is beneficial for promoting the differentiation of hMSCs towards the specific lineages of the resident cells in those particular tissues, facilitating their potential applications in musculoskeletal tissue engineering.