Human pluripotent stem cells (hPSCs) are a promising cell source with pluripotency and capacity to differentiate into all human somatic cell types. Designing simple and safe biomaterials with an innate ability to induce osteoblastic lineage from hPSCs is desirable to realize their clinical adoption in bone regenerative medicine. To address the issue, here we developed a fully defined synthetic peptides-decorated two-dimensional (2D) microenvironment via polydopamine (pDA) chemistry and subsequent carboxymethyl chitosan (CMC) grafting to enhance the culture and osteogenic potential of hPSCs in vitro. The hPSCs including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were successfully cultured on the peptides-decorated surface without Matrigel and ECM protein coating and underwent promoted osteogenic differentiation in vitro, determined from the alkaline phosphate (ALP) activity, gene expression, and protein production as well as calcium deposit amount. It was found that directed osteogenic differentiation of hPSCs was achieved through a peptides-decorated niche. This chemically defined and safe 2D microenvironment, which facilitates proliferation and osteo-differentiation of hPSCs, not only helps to accelerate the translational perspectives of hPSCs but also provides tissue-specific functions such as directing stem cell differentiation commitment, having great potential in bone tissue engineering and opening new avenues for bone regenerative medicine.