We present methods to characterize mesenchymal stromal cells (MSC) over long time periods in vitro. The methods entail passaging cells multiple times and performing differentiation studies with the cells at each passage. Using an array of surface markers and flow cytometric quantification, the data can be correlated to traditional measures of differentiation such as PCR and staining. Using these methods to quantify the amount of differentiation, we concluded that many common MSC markers do not specifically define MSCs with true stem cell properties. Additionally, adipose-derived as opposed to bone marrow-derived MSCs show long-term CD34⁺ labeling. The methods described can be used to help identify stem cell markers and to characterize the state of stem cells in vitro. Compiling these data from multiple laboratories would be helpful to determine source, extraction and culture methods needed to obtain high yields of useful stem cells.