Mesenchymal stem cells/multipotent stromal cells (MSCs) have a variety of unique properties that have made them a popular cell type in the study of tissue engineering. Their ability to undergo osteogenic, adipogenic and chondrogenic differentiation has long been appreciated. In addition to their capacity for skeletogenic differentiation, there are suggestions that MSCs have additional roles in organising tissue vasculature through interactions with endothelial cells (ECs). However, suitable experimental models to test these unique MSC activities are lacking and the mechanisms are unclear. Here, we have developed a novel 3D in vitro co-culture spheroid model of MSCs and ECs. Development and further investigations into this 3D co-culture spheroid model has resulted in many novel discoveries. Using calcium depletion from cell culture media to quantify osteogenic differentiation of MSCs in both 2D and 3D represents a complimentary assessment method that is non-destructive. Co-culture of MSCs and ECs was also found to promote osteogenic differentiation whilst having no detrimental effects on cell viability during long-term culture. Further investigations into the 3D co-culture of MSCs and ECs demonstrated spontaneous endothelial organisation. Using this model it was possible to track endothelial restructuring and identify the signalling processes involved, ultimately focusing on platelet-derived growth factor and notch signalling. Using a combination of pre-differentiated MSCs and ECs within the 3D co-culture system, osteochondral spheroids were developed. These spheroids were analysed using a combination of novel and traditional imaging techniques; it was found that osteochondral spheroids self-organised into distinct bone and cartilage regions, similar to those observed at the osteochondral boundary and during early endochondral ossification. This in vitro multiple cell culture system represent a simplified tractable model of osteochondral tissue.