Somatic Nucleophosmin (NPM1) mutation frequently occurs in acute myeloid leukemia (AML), but its role in leukemogenesis remains unclear. This study reports the first "conventional" knock-in mouse model of Npm1 mutation, which was achieved by inserting TCTG after nucleotide c.857 (c.854_857dupTCTG) to mimic human mutation without any "humanized" sequence. The resultant mutant peptide differed slightly different from that in humans but exhibited cytoplasmic pulling force. Homozygous (Npm1(c+/c+)) mice showed embryonic lethality before day E8.5, wheras heterozygous (Npm1(wt/c+)) mice appeared healthy at birth and were fertile. Approximately 36% of Npm1(wt/c+) mice developed myeloproliferative disease (MPD) with extramedullary hematopoiesis. Those Npm1(wt/c+) mice that did not develop MPD nevertheless gradually developed monocytosis and showed increased numbers of marrow myeloid precursors. This second group of Npm1(wt/c+) mice also showed compromised cobblestone area formation, suggesting pathology in the hematopoietic niche. Microarray experiments and bioinformatic analysis on mice myeloid precursor cells and 227 human samples revealed the expression of CXCR4/CXCL12-related genes was significantly suppressed in mutant cells from both mice and humans. Thus, our mouse model demonstrated that Npm1 mutation can result in MPD, but is insufficient for leukemogenesis. Perturbation of hematopoietic niche in mutant hematopoietic stem cells (implied by underrepresentation of CXCR4/CXCL12-related genes) may be important in the pathogenesis of NPM1 mutations.