We have previously reported that acute inducible knockout of the endoplasmic reticulum chaperone GRP94 led to an expansion of the hematopoietic stem and progenitor cell pool. Here, we investigated the effectors and mechanisms for this phenomenon. We observed an increase in AKT activation in freshly isolated GRP94-null HSC-enriched Lin(-) Sca-1(+) c-Kit(+) (LSK) cells, corresponding with higher production of PI(3,4,5)P3, indicative of PI3K activation. Treatment of GRP94-null LSK cells with the AKT inhibitor MK2206 compromised cell expansion, suggesting a causal relationship between elevated AKT activation and increased proliferation in GRP94-null HSCs. Microarray analysis demonstrated a 97% reduction in the expression of the hematopoietic cell cycle regulator Ms4a3 in the GRP94-null LSK cells, and real-time quantitative PCR confirmed this down-regulation in the LSK cells but not in the total bone marrow (BM). A further examination comparing freshly isolated BM LSK cells with spleen LSK cells, as well as BM LSK cells cultured in vitro, revealed specific down-regulation of Ms4a3 in freshly isolated BM GRP94-null LSK cells. On examining cell surface proteins that are known to regulate stem cell proliferation, we observed a reduced expression of cell surface connexin 32 (Cx32) plaques in GRP94-null LSK cells. However, suppression of Cx32 hemichannel activity in wild-type LSK cells through mimetic peptides did not lead to increased LSK cell proliferation in vitro. Two other important cell surface proteins that mediate HSC-niche interactions, specifically Tie2 and CXCR4, were not impaired by Grp94 deletion. Collectively, our study uncovers novel and unique roles of GRP94 in regulating HSC proliferation.