In order to investigate the influence of calcium and strontium ion concentration on human bone marrow stromal cells and their differentiation to osteoblasts, different cell culture media have been used. Even though this study does not contain a bone substitute material, the reason for this study was the decrease of cation concentration by many biomaterials, due to induced apatite precipitation. As a consequence, the reduced calcium ion concentration is known to affect osteoblastic development. Therefore, the main focus was put on the question, whether an increased strontium concentration (in the range of mM) might be suitable to compensate the lack of calcium ions. The effect of solely strontium ions—with only calcium in the media resulting from fetal calf serum—was investigated. Commercially available calcium-free medium (modified α-MEM) was tested in comparison with media with varied calcium ion concentrations (0.9, 1.8, and 3.6 mM), or strontium ion concentration (0.4, 0.9, 1.8, and 3.6 mM). In case of calcium, higher concentrations cause increased proliferation, while differentiation was shifted to earlier points of time. Differentiation was increased by solely strontium ions only at 0.4–0.9 mM, while proliferation was highest for 0.9–1.8 mM. From these results, it can be concluded that strontium is able to compensate a lack of calcium to a certain degree. Thus, in contrast to calcium ion release, a strontium ion release from bone substitute materials might be applicable for stimulation of bone regeneration without influencing the media saturation.