The transcription factors that regulate differentiation into the monocyte subset in bone marrow have not yet been identified. Here we found that the orphan nuclear receptor NR4A1 controlled the differentiation of Ly6C- monocytes. Ly6C- monocytes, which function in a surveillance role in circulation, were absent from Nr4a1-/- mice. Normal numbers of myeloid progenitor cells were present in Nr4a1-/- mice, which indicated that the defect occurred during later stages of monocyte development. The defect was cell intrinsic, as wild-type mice that received bone marrow from Nr4a1-/- mice developed fewer patrolling monocytes than did recipients of wild-type bone marrow. The Ly6C- monocytes remaining in the bone marrow of Nr4a1-/- mice were arrested in S phase of the cell cycle and underwent apoptosis. Thus, NR4A1 functions as a master regulator of the differentiation and survival of 'patrolling' Ly6C- monocytes.