Tendon injury occurs frequently and tendon repair is limited by its poor self-healing. The current tissue engineering approach for treating tendon injuries has showed limited success, largely due to the lack of scaffolds with suitable structural and biological properties, and suitable growth factors for differentiation of stem cells into tendon cells. This study investigated if the combination of environmental and biological cues from aligned chitosan-poly-caprolactone (C-PCL) combined with TGF-β3 growth factor can efficiently and rapidly direct the tenogenic differentiation of primary human bone marrow stem cells (BMSCs). C-PCL nanofibers were prepared to have the anisotropic nanostructure, and mechanical and biological properties similar to those of the native tendon extracellular matrix (ECM). The tenogenic commitment of BMSCs was assessed using cell morphology, and gene and protein expressions. BMSCs grown on uniaxially aligned C-PCL nanofibers in a medium containing TGF-β3 displayed an elongated morphology along nanofiber orientation, upregulated expressions of marker genes, and increased collagen production associated with tenogenic differentiation as compared to control substrates. Significantly, this tenogenic microenvironment induced the transcription of tenogenic markers in 5 days and production of a large amount of Collagen I in 10 days, more effective and faster than existing scaffolds combined with growth factors. This research reveals that a combinative effect of aligned C-PCL nanofibers and TGF-β3, as environmental and biological cues, can lead to rapid, effective BMSC differentiation into tenogenic progenitors, offering a potential strategy for managing tendon disorders.