Megakaryocytes are the precursor cells of platelets and are bona fide resident cells in the bone marrow but extremely low in numbers (~1% of total nucleated cells). Upon terminal differentiation, megakaryocytes increase their size, become polyploid and develop a demarcation membrane system. Mature megakaryocytes form proplatelets, which are cytoplasmic extensions that protrude through the endothelial cell layer of venous sinusoids within the bone marrow, entering into the blood circulation and, subsequently, releasing platelets. Despite limited in numbers, megakaryocytes have been successfully isolated from bone marrow (Tolhurst et al., 2012), adult peripheral blood (Mazur et al., 1990; Thornton et al., 1999), cord blood (Sun et al., 2004) and also from embryonic stem cells (Pick et al., 2013; Eto et al., 2002). These procedures rely on immunostaining using antibodies against megakaryocyte surface markers (i.e. CD41 or CD42b) to isolate an enriched population of megakaryocytes. Here, we describe a culture method wherein megakaryocytes can be grown and differentiated in vitro from human peripheral blood mononuclear cells (PBMCs) directly without the need of initial isolation of CD34+ cells. This method is based on a previously published culture method of human erythroid progenitor cells from PBMCs (Borg et al., 2010; Leberbauer et al., 2005). Although the purity of megakaryocytes is not 100% in this culture method, an enriched fraction of megakaryocytes can be further isolated using BSA gradient or cell-sorting techniques. In addition, our method offers the possibility to freeze the cultures after minimal expansion of yet undifferentiated megakaryocytes, which will yield equal megakaryocyte cultures after thawing when compared to fresh uninterrupted cultures. As this has been proven difficult with CD34+ sorted pluripotent cells, it allows managing samples and to perform downstream analysis when human material is not always available.