In recent years, multipotent mesenchymal stromal cells (MSCs) have demonstrated tremendous potential for use in regenerative medicine. CXCR4, the receptor for CXCL12, is highly expressed by bone marrow (BM) MSCs and the CXCR4/CXCL12 axis has been shown to be important for migration and homing of BM-MSCs. Typically, MSCs used for clinical applications are collected after culture expansion using enzymatic methods, such as trypsin. Here, we compared different commercially available enzymatic and non-enzymatic methods for collection and dissociation of MSCs from culture plastics and their effects on CXCR4 expression by MSCs. We found that whereas non-enzymatic dissociation buffers and methods maintained CXCR4 expression, all tested enzymatic dissociation solutions dramatically decreased expression of CXCR4. We, therefore, strongly recommend the use of non-enzymatic dissociation methods, followed by filtration through a cell strainer to obtain single cell suspensions, in order to preserve maximal CXCR4 expression and optimal homing of cells.