Bone tissue engineering using human mesenchymal stem cells (hMSCs) is a multidisciplinary field that aims to treat patients with trauma, spinal fusion and large bone defects. Cell-based bone tissue engineering encompasses the isolation of multipotent hMSCs from the bone marrow of the patient, in vitro expansion and seeding onto porous scaffold materials. In vitro pre-differentiation of hMSCs into the osteogenic lineage augments their in vivo bone forming capacity. Differentiation of hMSCs into bone forming osteoblasts is a multi-step process regulated by various molecular signaling pathways, which warrants a thorough understanding of these signaling cues for the efficient use of hMSCs in bone tissue engineering. Recently, there has been a surge of knowledge on the molecular cues regulating osteogenic differentiation but extrapolation to hMSC differentiation is not guaranteed, because of species- and cell-type specificity. In this review, we describe a number of key osteogenic signaling pathways, which directly or indirectly regulate osteogenic differentiation of hMSCs. We will discuss how and to what extent the process is different from that in other cell types with special emphasis on applications in bone tissue engineering.