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Summary We describe a protocol for a long-term co-culture assay to study the contribution of mesenchymal stromal cells (MSCs) in regulating hematopoietic stem/progenitor cell (HSPC) activity. In addition, we describe the use of a clonogenic assay to determine myelo-erythroid differentiation. This long-term culture-initiating cell assay can be used for qualitative analysis of MSCs capable of supporting hematopoiesis and may also be used as a proxy readout to study HSPC repopulation. For complete details on the use and execution of this protocol, please refer to Sinha et al. (2020)., Graphical Abstract, Highlights • We report long-term co-culture of mesenchymal stroma and hematopoietic stem/progenitors • End-point colony-forming analysis helps determine myelo-erythroid differentiation • This protocol analyzes mesenchymal stromal cell potential to support hematopoiesis • Long-term culture-initiating cell assay is a surrogate for hematopoietic engraftment, We describe a protocol for a long-term co-culture assay to study the contribution of mesenchymal stromal cells (MSCs) in regulating hematopoietic stem/progenitor cell (HSPC) activity. In addition, we describe the use of a clonogenic assay to determine myelo-erythroid differentiation. This long-term culture-initiating cell assay can be used for qualitative analysis of MSCs capable of supporting hematopoiesis and may also be used as a proxy readout to study HSPC repopulation.