Hematopoiesis is regulated by the bone marrow (BM) niche microenvironment. We recently found that post transplant administration of AMD3100 (a specific and reversible CXCR4 antagonist) enhanced donor cell engraftment and promoted recovery of all donor cell lineages in a congeneic mouse transplant model. We hypothesized that AMD3100 enhances donor cell reconstitution in part by modulating the levels and constitution of soluble factors in the niche microenvironment. In the current study, the effects of the BM extracellular fluid (supernatant) from AMD3100-treated transplant recipient mice on colony forming units (CFUs) were examined. A semi-quantitative, mass spectrometry-based proteomics approach was used to screen for differentially expressed proteins between the BM supernatants of PBS-treated transplant mice and AMD3100-treated transplant mice. A total of 178 proteins were identified in the BM supernatants. Thioredoxin was among the 32 proteins that displayed >2-fold increase in spectral counts in the BM supernatant of AMD3100-treated transplant mice. We found that thioredoxin increased CFUs in a dose-dependent manner. Thioredoxin improved hematopoiesis in irradiated mice and protected mice from radiation-related death. Furthermore, ex vivo exposure to thioredoxin for 24 hours enhanced the long-term repopulation of hematopoietic stem cells. Additionally, combined post transplant administration of thioredoxin and AMD3100 improved hematological recovery in primary and secondary transplant recipient mice. Our studies demonstrated that factors in the BM niche microenvironment play a critical role in hematopoiesis. Identifying these factors provides clues on potential novel targets that can be used to enhance hematological recovery in hematopoietic stem cell transplantation.