Despite increasing knowledge on the regulation of hematopoietic stem/progenitor cell (HSPC) self-renewal and differentiation, in vitro control of stem cell fate decisions has been difficult. The ability to inhibit HSPC commitment in culture may be of benefit to cell therapy protocols. Small molecules can serve as tools to manipulate cell fate decisions. Here, we tested 2 small molecules, valproic acid (VPA) and lithium (Li), to inhibit differentiation. HSPCs exposed to VPA and Li during differentiation-inducing culture preserved an immature cell phenotype, provided radioprotection to lethally irradiated recipients, and enhanced in vivo repopulating potential. Anti-differentiation effects of VPA and Li were observed also at the level of committed progenitors, where VPA re-activated replating activity of common myeloid progenitor and granulocyte macrophage progenitor cells. Furthermore, VPA and Li synergistically preserved expression of stem cell-related genes and repressed genes involved in differentiation. Target genes were collectively co-regulated during normal hematopoietic differentiation. In addition, transcription factor networks were identified as possible primary regulators. Our results show that the combination of VPA and Li potently delays differentiation at the biologic and molecular levels and provide evidence to suggest that combinatorial screening of chemical compounds may uncover possible additive/synergistic effects to modulate stem cell fate decisions.