We identified nicotinamide phosphoribosyltransferase (NAMPT), also known as pre-B cell colony enhancing factor (PBEF), as an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis in healthy individuals and in individuals with severe congenital neutropenia. Intracellular NAMPT and NAD(+) amounts in myeloid cells, as well as plasma NAMPT and NAD(+) levels, were increased by G-CSF treatment of both healthy volunteers and individuals with congenital neutropenia. NAMPT administered both extracellularly and intracellularly induced granulocytic differentiation of CD34(+) hematopoietic progenitor cells and of the promyelocytic leukemia cell line HL-60. Treatment of healthy individuals with high doses of vitamin B3 (nicotinamide), a substrate of NAMPT, induced neutrophilic granulocyte differentiation. The molecular events triggered by NAMPT include NAD(+)-dependent sirtuin-1 activation, subsequent induction of CCAAT/enhancer binding protein-alpha and CCAAT/enhancer binding protein-beta, and, ultimately, upregulation of G-CSF synthesis and G-CSF receptor expression. G-CSF, in turn, further increases NAMPT levels. These results reveal a decisive role of the NAD(+) metabolic pathway in G-CSF-triggered myelopoiesis.