Hematopoiesis is maintained throughout life by self-renewing hematopoietic stem cells (HSCs) that differentiate to produce both myeloid and lymphoid cells. The NR4A family of orphan nuclear receptors, which regulates cell fate in many tissues, appears to play a key role in HSC proliferation and differentiation. Using a NR4A1^GFP BAC transgenic reporter mouse we have investigated NR4A1 expression and its regulation in early hematopoiesis. We show that NR4A1 is most highly expressed in a subset of Lin⁻Sca-1⁺c-Kit⁺ CD48⁻CD150⁺ long-term (LT) HSCs, and its expression is tightly associated with HSC quiescence. We also show that NR4A1 expression in HSCs is induced by PGE₂, a known enhancer of stem cell engraftment potential. Finally, we find that both NR4A1^GFP+ and NR4A1^GFP− HSCs successfully engraft primary and secondary irradiated hosts; however, NR4A1^GFP+ HSCs are distinctly myeloid-biased. These results show that NR4A1 expression identifies a highly quiescent and distinct population of myeloid-biased LT-HSCs. Stem Cells  2015;33:278–288