Mesenchymal stem cells (MSCs) are crucial cells that play an essential role in the maintenance, self‐renewal, and proliferation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow niche. It has been proven that MSCs can be used as a feeder layer for the proliferation of HSCs to enhance the number of HPCs and HSCs. Recently, it has been demonstrated that MSC‐derived exosome (MSC‐DE) has critical roles in different biological processes in bone marrow (BM). In the current research, we examined the importance of hypoxia‐preconditioned MSC‐derived exosomes (HP‐MSC‐DE) and normoxia‐preconditioned MSC‐derived exosomes (NP‐MSC‐DE) in the self‐renewal and long‐term clonogenic potential of umbilical cord blood hematopoietic stem cells (UCB‐HSCs). We showed that the secretion rate and component of the exosome (EXO) were changed in HP‐MSC‐DE compared to NP‐MSC‐DE. Notably, the Jagged‐1 (Notch ligand) content of EXO was much more plentiful in HP‐MSC‐DE compared to NP‐MSC‐DE. The addition of HP‐MSC‐DE enriched by Jagged‐1 to the co‐culture system stimulates the Notch pathway on the membrane of UCB‐HSCs CD133+ and enhances proliferation. HP‐MSC‐DE induction using an anti‐Jagged‐1 antibody suppresses all biological functions of the Jagged‐1 protein. Importantly, HP‐MSC‐DE containing Jagged‐1 could change the biology of HSCs CD133+ and increase the self‐renewal capacity, quiescence, and clonogenic potential of CD133+ cells. Moreover, they support generating a large number of primitive cells. Our study signified the importance of HP‐MSC‐DE in the proliferation of UCB‐HSCs CD133+, which manifested therapeutic applications of EXO in the enhanced number of HSCs and subsequently alleviated bone marrow transplantation.