Cartilage engineering with stem cells in 3D scaffolds is a promising future therapy to treat cartilage defects. One challenge in the field is to design carriers to efficaciously deliver biological factors in 3D scaffolds containing stem cells to appropriately guide differentiation of these cells in same scaffolds and promote specific tissue synthesis. Graphene-based 2D nanomaterials have recently attracted extensive interest for their biomedical applications as they can adsorb a plethora of biological molecules, thus offering high potential as delivery carriers. This study utilized graphene oxide (GO) flakes to adsorb transforming growth factor β3 (TGF-β3), which were then incorporated into a collagen hydrogel. Human mesenchymal stem cells (hMSCs) were encapsulated in the same gel and chondrogenic differentiation assessed. The study showed GO flakes adsorbed > 99% TGF-β3 with <1.7% release. Adsorbed TGF-β3 retained a similar conformation to its dissolved counterpart (free protein) but importantly demonstrated greater conformational stability. Smad2 phosphorylation was promoted, and higher chondrogenic gene expression and cartilage-specific extracellular matrix deposition were achieved compared to exogenously delivering TGF-β3 in culture media. Effects were sustained in long-term 28-day culture. The results demonstrate GO flakes as highly-efficient for delivering GFs in 3D to guide cells in the same scaffold and induce tissue formation. The ability of GO flakes to provide sustained local delivery makes this material attractive for tissue engineering strategies, in particular for regionally-specific MSC differentiation (e.g. osteochondral tissue engineering). STATEMENT OF SIGNIFICANCE: Cartilage engineering involving stem cells in 3D scaffolds is a promising future therapy to treat cartilage defects which can lead to debilitating conditions such as osteoarthritis. However, this field faces the challenge to design delivery carriers to efficaciously deliver biological factors inside these 3D cell-containing scaffolds for appropriately-guided cell differentiation. Graphene-based 2D nanomaterials offer high potential as delivery carriers, but to date studies using them to deliver biological factors have been restricted to 2D substrates, non-scaffold cell masses, or acellular 3D scaffolds. Our study for the first time demonstrated simultaneously incorporating both human mesenchymal stem cells (hMSCs) and GO (graphene oxide)-adsorbed growth factor TGFβ3 into a 3D scaffold, where GO-adsorbed TGFβ3 enhanced chondrogenic differentiation of hMSCs and cartilage-tissue synthesis throughout the scaffold without needing to repeatedly supply TGFβ3 exogenously.