Electrospun scaffolds are potentially interesting in bone tissue engineering due to a strong structural similarity to the natural bone matrix. To investigate the osteogenic behavior of cells on the scaffolds, dynamic culture of cells is essential to simulate the biological environment. In the present study, human mesenchymal stem cells (hMSCs) were cultured on multilayer nanohydroxyapatite-polycaprolactone electrospun scaffolds at different configurations (horizontal with or without pressure and parallel with the medium flow) and flow rates in a perfusion bioreactor. Alkaline phosphatase (ALP) activity, cell viability, Ca deposition and RUNX2 expression were determined in three different dynamic states, and compared with static culture after 1, 3, 7, and 14 days. Among dynamic groups, RUNX2 gene expression upregulated more in a horizontal state at a low flow rate without mechanical pressure (LF) and parallel flow (PF), than static group on day 7. At a high flow rate with mechanical pressure, Ca deposition and ALP activity increased 2.34 and 1.7 folds more than in static culture over 7 days, respectively. Furthermore, ALP activity, Ca deposition and RUNX2 gene expression increased in PF samples. PF provided longer culture time with higher cell differentiation. Therefore, high flow rate with mechanical pressure and PF are suggested for producing differentiated cell structure for bone tissue engineering.