To date, different experimental strategies have been developed for the ex vivo expansion of human hematopoietic stem (HSCs) and progenitor (HPCs) cells. This has resulted in significant advances on the use of such expanded cells in transplantation settings. To this day, however, it is still unclear to what extent those stem and progenitor cells generated in vitro retain the functional and genomic integrity of their freshly isolated counterparts. In trying to contribute to the solving of this issue, in the present study we have selected and purified three different hematopoietic cell populations: HSCs (CD34+ CD38‐ CD45RA‐ CD71‐ Lin‐ cells), myeloid progenitor cells (CD34+ CD38+ CD45RA+ CD71‐ Lin‐ cells), and erythroid progenitor cells (CD34+ CD38+ CD45RA‐ CD71+ Lin‐ cells), obtained directly from fresh human umbilical cord blood (UCB) units or generated in vitro under particular culture conditions. We, then, compared their functional integrity in vitro and their gene expression profiles. Our results indicate that in spite of being immunophenotipically similar, fresh and in vitro generated cells showed significant differences, both in functional and genetic terms. As compared to their fresh counterparts, those HSCs generated in our culture system showed a deficient content of long‐term culture‐initiating cells, and a marked differentiation bias toward the myeloid lineage. In addition, in vitro generated HSCs and HPCs showed a limited expansion potential. Such functional alterations correlated with differences in their gene expression profiles. These observations are relevant in terms of HSC biology and may have implications in UCB expansion and transplantation. Stem Cells Translational Medicine 2018;7:602–614