Biophysical stimulation regulates stem cell functions, including proliferation and differentiation. Matrix nanotopography and external forces, such as electromagnetic fields (EMF), can enhance this stimulation. Here, it is demonstrated that biophysical multiple cues coordinated from electromagnetized Au-nanoparticles-decorated polymer nanofiber under EMF significantly regulate the adhesion, alignment, proliferation, and lineage commitment of hMSCs. Without EMF, matrix cues of electrical conductivity and nanodotted fibrous topography accelerate the anchorage and spreading of hMSCs. Of note, EMF synergizes with the matrix cues to enhance cellular behaviors, resulting in elongated and aligned cells along the field direction. Microtubules are highly polymerized, acetylated, and aligned, playing an active role in these events. Actin filaments also develop in parallel with the microtubules, facilitating actin-microtubule crosstalks. These phenomena lead to changes in the nuclear mechanics of hMSCs, including elongated nuclear shape and decondensed chromatins with histone acetylation. The EMF+matrix-stimulated hMSCs express genes related to microtubule organization and euchromatin, as revealed by RNA sequencing, and show chromatin accessibility with enrichment of genes related to mechanotransduction and lineage specification, as analyzed by ATAC sequencing. The EMF+matrix biophysical stimulation further increases the capacity for lineage specification (predominantly towards osteogenic, myogenic, and tenogenic), offering a promising bioengineering platform for stem cell engineering and therapies.