The stemness of ex vivo expanded hematopoietic stem cells (HSCs) is usually compromised by current methods. To explore the failure mechanism of stemness maintenance of human HSCs, which were expanded from human umbilical cord blood (hUCB) CD34(+)cells, by differentiation inhibitor Stem Regenin 1 (SR1), an antagonist of aryl hydrocarbon receptor, we investigated the activity of p38 mitogen-activated protein kinase alpha (p38 MAPK alpha, p38 alpha) and mammalian target of rapamycin complex 1 (mTORC1), and their effect on SR1-expanded hUCB CD34(+)cells. Our results showed that cellular senescence occurred in the SR1-expanded hUCB CD34(+)cells in which p38 alpha and mTORC1 were successively activated. Furthermore, their coinhibition resulted in a further decrease in hUCB CD34(+)cell senescence without an effect on apoptosis, promoted the maintenance of expanded phenotypic HSCs without differentiation inhibition, increased the hematopoietic reconstitution ability of multiple lineages, and potentiated the long-term self-renewal capability of HSCs from SR1-expanded hUCB CD34(+)cells in NOD/Shi-scid/IL-2R gamma(null)mice. Our mechanistic study revealed that senescence inhibition by our strategy was mainly attributed to downregulation of the splicesome, proteasome formation, and pyrimidine metabolism signaling pathways. These results suggest that coinhibition of activated p38 alpha and mTORC1 potentiates stemness maintenance of HSCs from SR1-expanded hUCB CD34(+)cells via senescence inhibition. Thus, we established a new strategy to maintain the stemness of ex vivo differentiation inhibitor-expanded human HSCs via coinhibition of multiple independent senescence initiating signal pathways. This senescence inhibition-induced stemness maintenance of ex vivo expanded HSCs could also have an important role in other HSC expansion systems.