Objective The number of hematopoietic stem and progenitor cells (HPCs) per cord blood unit is limited, and this can result in delayed engraftment or graft failure. In vitro expansion of HPCs provides a perspective to overcome these limitations. Cytokines as well as mesenchymal stromal cells (MSCs) have been shown to support HPCs ex vivo expansion, but a systematic analysis of their interplay remains elusive. Materials and Methods Twenty different combinations of growth factors (stem cell factor [SCF], thrombopoietin [TPO], fibroblast growth factor−1 [FGF-1], angiopoietin-like 5, and insulin-like growth factor-binding protein 2), either with or without MSC coculture were systematically compared for their ability to support HPC expansion. CD34 + cells were stained with carboxyfluorescein diacetate N-succinimidyl ester to monitor cell division history in conjunction with immunophenotype. Colony-forming unit frequencies and hematopoietic reconstitution of nonobese diabetic severe combined immunodeficient mice were also assessed. Results Proliferation of HPCs was stimulated by coculture with MSCs. This was further enhanced in combination with SCF, TPO, and FGF-1. Moreover, these conditions maintained expression of primitive surface markers for more than four cell divisions. Colony-forming unit−initiating cells were not expanded without stromal support, whereas an eightfold increase was reached by simultaneous cytokine-treatment and MSC coculture. Importantly, in comparison to expansion without stromal support, coculture with MSCs significantly enhanced hematopoietic chimerism in a murine transplantation model. Conclusions The supportive effect of MSCs on hematopoiesis can be significantly increased by addition of specific recombinant growth factors; especially in combination with SCF, TPO, and FGF-1.