Human adipose-derived stem cells (hASCs) are an attractive cell source for stem cell research and regenerative medicine. Being capable to undergo osteogenic differentiation,however, the involved molecular pathways are poorly understood. Here, we focused on lineage commitment in dependence of different hASC donors via inhibiting the mitogen-activated protein kinases ERK and JNK, and their regulators FAK and Ras. Differentiation was analyzed under control, osteogenic, and solvent DMSO conditions with respect to cell morphology, focal adhesion formation, extracellular matrix production, expression of the transcription factors Runx2 and osterix, BMP-2- Smad and ERK signaling, and osteogenic markers (alkaline phosphatase activity, calcium mineralization, osteocalcin/osteopontin expression). Under control conditions, no osteogenic differentiation occurred. After inhibition the extent of each parameter varied with the hASC donor; the solvent had no remarkable impact on osteogenic markers compared with the osteogenic differentiation. The inhibitors of FAK and JNK cross talked with ERK and reduced the investigated parameters the strongest. Inactivation of ERK caused a moderate and marker specific inhibition. Blocked Ras promoted the cell responses in an ERK-independent way. All pathways depend on Runx2 and osterix. A correlation between the interplay with BMP-2-Smad pathway, and cell elongation with osteogenic differentiation could not be determined, while focal adhesion formation was essential.