Abstract The important contributions of the α4 integrin VLA-4 and the CXCR4/SDF-1 axis in mobilization have been demonstrated and thereby, these pathways can be suggested as rational targets for clinical stem cell mobilization in the absence of cytokine use. α4-blockade alone (in humans, macaques and mice), or genetic ablation of α4-integrin in mice, provides reproducible, but modest mobilization. Similarly, CXCR4 blockade with small-molecule antagonists mobilizes hematopoietic stem cells in all three species, but at least with the established single-injection schedule, the mobilization efficiency is marginally sufficient for clinical purposes. Hypothesizing that the different molecular targets (α4-integrin vs. CXCR4) might allow for additive mobilization effects, we therefore tested the efficacy of the combination of α4-integrin blockade with anti-functional antibodies and CXCR4 blockade with the small-molecule inhibitor AMD3100 in macaques, or the combination of conditional α4-integrin ablation and AMD3100 in mice. Mobilization was at least additive. While the prolonged effects of α4-blocking antibodies may not be suitable for clinical mobilization, future availability of small-molecule α4-antagonists in combination with AMD3100 could provide an alternative to granulocyte colony-stimulating factor. Disclosure of potential conflicts of interest is found at the end of this article.