Establishment of a defined cell culture system that facilitates ex vivo expansion of isolated hematopoietic stem and progenitor cells (HSPCs) is a crucial issue in hematology and stem cell transplantation. Here we have evaluated the capacity of primary human multipotent mesenchymal stromal cells (MSCs) to support the ex vivo expansion of peripheral CD34(+)-enriched HSPCs. We observed that HSPCs co-cultured on MSCs showed a substantially higher total expansion rate compared to those growing without. Moreover, in addition to the expansion of CD34(+)CD133(+) and CD34(+)CD133(-) cells, a third population of CD133(+)CD34(-) stem cells became detectable after expansion. Direct contact between HSPCs and the feeder layer appears beneficial for the expansion of HSPCs harboring CD133(+) phenotype, i.e., CD34(+)CD133(+) and CD133(+)CD34(-), in contrast to CD34(+)CD133(-) cells. Interestingly, electron microscopy and immunofluorescence analyses revealed that adherent HSPCs display various morphologies; they are either round with, in some cases, the appearance of a microvillar pole or exhibit several distinct types of plasma membrane protrusions such as lamellipodium and magnupodium. CD133 is selectively concentrated therein, whereas CD34 is randomly distributed over the entire surface of HSPCs. Together, this co-culture offers a unique experimental system to further characterize the biology and role of markers of rare stem cell populations.