Hematopoiesis is orchestrated by interactions between hematopoietic stem/progenitor cells (HSPCs) and stromal cells within bone marrow (BM) niches. Side population (SP) functionality is a major characteristic of HSPCs related to quiescence and resistance to drugs and environmental stresses. At steady state, SP cells are mainly present in the BM and are mostly absent from the circulation except in stress conditions, raising the hypothesis of the versatility of the SP functionality. However, the mechanism of SP phenotype regulation is unclear. Here we show for the first time that the SP functionality can be induced in lin(-) cells from unmobilized peripheral blood after nesting on mesenchymal stromal cells (MSCs). This MSC-induced SP fraction contains HSPCs as demonstrated by their (i) CD34(+) cell percentage, (ii) quiescent status, (iii) in vitro proliferative and clonogenic potential, (iv) engraftment in NSG (NOD SCID gamma chain) mice and (v) stemness gene expression profile. We demonstrate that SP phenotype acquisition/reactivation by circulating lin(-) cells is dependent on interactions with MSCs through VLA-4/α4β1-integrin and CD44. A similar integrin-dependent mechanism of SP phenotype acquisition in acute myeloid leukemia circulating blasts suggests an extrinsic regulation of ATP-binding cassette-transporter activity that could be of importance for a better understanding of adhesion-mediated chemoresistance mechanisms.