Electrospun scaffolds provide a promising approach for tissue engineering as they mimic the physical properties of extracellular matrix. Previous studies have demonstrated that electrospun scaffolds with porous features on the surface of single fibers, enhanced cellular attachment and proliferation. Yet, little is known about the effect of such topographical cues on cellular differentiation. Here, we aimed at investigating the influence of surface roughness of electrospun scaffolds on skeletal differentiation of human mesenchymal stromal cells (hMSCs). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) analysis showed that the surface nanoroughness of fibers was successfully regulated via humidity control of the electrospinning environment. Gene expression analysis revealed that a higher surface roughness (roughness average (Ra) = 71.0 ± 11.0 nm) supported more induction of osteogenic genes such as osteopontin (OPN), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2), while a lower surface roughness (Ra = 14.3 ± 2.5 nm) demonstrated higher expression of other osteogenic genes including bone sialoprotein (BSP), collagen type I (COL1A1) and osteocalcin (OCN). Interestingly, a lower surface roughness (Ra = 14.3 ± 2.5 nm) better supported chondrogenic gene expression of hMSCs at day 7 compared to higher surface roughness (Ra = 71.0 ± 11.0 nm). Taken together, modulating surface roughness of 3D scaffolds appears to be a significant factor in scaffold design for the control of skeletal differentiation of hMSCs.