Mesenchymal stem cells (MSCs) can transfer hematopoietic microenvironment in vivo. After implantation of murine femur bone marrow (BM) plug or adherent cell layer (ACL) of long-term bone marrow culture (LTBMC) established from the same BM equivalent, ectopic hematopoietic focus is formed under the renal capsule of syngeneic recipient. Stromal cells in those foci are derived from donor MSCs, while hematopoietic cells are of recipient origin. Cellularity of individual foci is proportional to the number of transplanted femur equivalents. Consequently, this assay can be used to quantitatively assess the number of MSCs in BM. Based on the self-renewal potential of MSCs, ectopic foci can be serially retransplanted. The aim of this study was to characterize the clonal structure of MSC populations. LTBMCs were obtained from F1 (CBA x C57Black/J6) mice. Cultures were transduced 2 weeks after establishment with a lentiviral library containing genetic barcodes (32 variable nucleotides). After additional 2 weeks, the proportion of marked stromal clonogenic cells (colony forming units fibroblast /CFU-F/) in ACLs was 73.7±5.1%. ACLs were implanted under the renal capsules of syngeneic recipients. Six weeks later the developed ectopic foci were partially retransplanted. DNA was isolated from LTBMCs, initial and secondary ectopic foci. Barcode sequences were determined on Illumina platform. In initial ectopic foci 278±40 (n = 7) individual barcodes were identified. The secondary ectopic foci contained 247±23 (n = 4) barcodes. Only 12±1 common barcodes were found in both initial and secondary foci. Since the secondary were descendants of the initial foci, we calculated that the initial foci contained about 500 barcoded MSCs, meaning that at least 2.3% of MSCs took part in the formation of hematopoietic microenvironment in the initial and the secondary foci. All other MSCs formed microenvironment only once. Taking into account the proportion of marked stromal clonogenic cells in ACLs, initial foci contained at least 600 MSCs. As each LTBMC was established from one femur, a femur contains no less than 600 MSCs. Presuming a linear relationship between the number of implanted MSCs and the size of ectopic foci, our data suggest that not all MSCs, but only part of them is involved in formation of hematopoietic microenvironment.