Megakaryocytes are the main members of the hematopoietic system responsible for regulating vascular homeostasis through their progeny platelets, which are generally known for maintaining hemostasis. Megakaryocytes are characterized as large polyploid cells that reside in the bone marrow but may also circulate in the vasculature. They are generated directly or through a multi-lineage commitment step from the most primitive progenitor or Hematopoietic Stem Cells (HSCs) in a process called “megakaryopoiesis”. Immature megakaryocytes enter a complicated development process defined as “thrombopoiesis” that ultimately results in the release of extended protrusions called proplatelets into bone marrow sinusoidal or lung microvessels. One of the main mediators that play an important modulatory role in hematopoiesis and hemostasis is nitric oxide (NO), a free radical gas produced by three isoforms of nitric oxide synthase within the mammalian cells. In this review, we summarize the effect of NO and its signaling on megakaryopoiesis and thrombopoiesis under both physiological and pathophysiological conditions.