The biological, chemical, and mechanical properties of the extracellular matrix (ECM) are important for adhesion, proliferation, and osteogenic differentiation of mesenchymal stromal cells (MSC). Scaffolds prepared for tissue engineering approaches should imitate the properties of the native ECM of the target tissue. Here, we used the synthetic hydrogel HyStem™-HP with different elasticity as a substrate for human bone marrow derived MSC (hBMMSC) and determined the influence of elasticity on morphology, adhesion, proliferation, and osteogenic differentiation of the cells. hBMMSC cultured on HyStem™-HP with a crosslinking of 1.6% (low elasticity) were well-spread, revealed an organized actin cytoskeleton and many focal adhesion (FA) contacts in comparison cells cultured on HyStem™-HP with a crosslinking of 0.1% (high elasticity) showed less spreading, less FAs, and a less organized actin cytoskeleton. Following osteogenic differentiation markers, like the activity of tissue-non-specific alkaline phosphatase, bsp II expression, and calcium accumulation were more pronounced on HyStem™-HP 1.6% hydrogels compared to HyStem™-HP 0.1%. These findings indicate that osteogenic differentiation of hBMMSC is better promoted by HyStem™-HP with low elasticity and might therefore be a useful substrate for bone tissue engineering.