Mesenchymal stem cells (MSCs) are capable of differentiating into, as well as contributing to the regeneration of mesenchymal tissues such as bone, cartilage, muscle and adipose tissues. In an effort to develop an in vitro assay that better mimics developmental and maintenance pathways known to drive osteogenesis in vivo, we investigated the synergistic effects of canonical Wnt and BMP signalling. Importantly, we aimed also to remove dexamethasone, a synthetic glucocorticoid common to all osteogenic medias, but that is absent in vivo. The results of our investigation confirm that activating Wnt signalling alone is not sufficient to induce osteogenesis, but that simultaneous activation of both Wnt and BMP pathways combined with optimisation of the base media components can significantly improve the differentiation outcome in vitro by at least 6-fold, when compared to standard osteogenic conditions. Long-term (7 days) activation induces early hydroxyapatite formation (by day 11), with increased cell apoptosis, and even observation of entombment of live cells within mineralised matrix by day 14. On the contrary, short-term activation (4 days) also leads to hydroxyapatite formation, but it can be seen only around day 14, with little or no apoptosis. Using our completely defined media, both in terms of components and pathway stimulation, our results confirm that hMSCs can be differentiated into osteoblasts in a more defined manner that is aligned with in vivo scenarios, instead of using a cocktail of factors with ambiguous pathway activation.