Bone marrow mesenchymal stromal cells (MSCs) play an essential role in the regulation of normal and leukemic hematopoiesis. Their multipotent potential of differentiation also makes them an interesting therapeutic tool. Among factors involved in the regulation of MSCs, energy metabolism plays a key role in their proliferation and differentiation. Seahorse Bioscience introduced extracellular flux technology to the life sciences market in 2006. This methodology allows, in living cells and in real time, the concomitant determination of basal oxygen consumption, glycolysis rates, ATP production, and respiratory capacity in a single experiment. Here we describe the protocol used to study concomitantly the respiratory and glycolytic metabolism of primary MSCs from the determination of oxygen consumption (OCR) and extracellular acidification (ECAR) rates.