The generation of platelets and megakaryocytes (MK) in vitro provides a potent strategy to overcome the shortage of today’s platelet transfusions to patients with acute needs. The amount of patients, depending on donations, and in need for transfusions are increasing while platelet donors are not more frequent. Furthermore, the risk of bacterial contamination in platelet products is high due to their storage at room temperature to prevent platelet activation. This also shortens the shelf life of platelet products. After repetitive transfusions, patients can develop refractoriness and immunity adding further complications to the use of platelet therapies. Therefore, alternative sources for platelets are sought for. Platelets can be produced in vitro from MK progenitors, hematopoietic stem cells or pluripotent stem cells. However, production is inefficient, particularly taking in consideration that the tremendous amount 4-5 x 1011 platelets have to be produced for filling one transfusion unit. In this study, we wanted to explore whether the expression of single supporting candidate factors in murine induced pluripotent stem cells (iPSC) could increase platelet production in vitro. We concentrated on the overexpression of major transcription factors of earlyphase megakaryopoiesis (Gata1, Pbx1, and Evi1) and late phase (Nfe2) to enhance MK production platelet release. Furthermore, to support platelet release from MK, we expressed hyperactive variants of the small GTPases RhoA and Cdc42. Gene modifications in iPSC, however, experience major drawbacks because of silencing of ectopic gene expression especially during differentiation. Furthermore, expression of the supportive factors has to be tightly controlled to avoid unwanted interference of the factors with pluripotency and differentiation since the transduction has to be performed in the pluripotent state. To overcome this, a tet-inducible expression system in iPSC was developed which was based on the generation of iPSC containing the reverse tetracycline (rt) transactivator TA-M2 in the Rosa26 safe harbor locus to prevent gene silencing of the (rt)TA-M2 and thereby guaranteeing its robust expression throughout the differentiation process. After verification of pluripotency and differentiation potential of the generated iPSC, the candidate factors were introduced with gammaretroviral vectors containing the respective transgene expressed from a Tet-responsive minimal promoter. In our experiments we found that overexpression of Gata1 elevated production of megakaryocyte progenitors, however, full maturation of the cells was impaired. In contrast, overexpression of Nfe2 elevated numbers of released platelets. In a second approach, we developed a doxycycline (dox)-induced overexpression system that should be transferable to human cells. We improved Tet-inducible all-in-one gammaretroviral vectors by introducing different ubiquitous chromatin opening elements derived from the human HNRPA2B1/CBX3 locus (A2UCOE), previously proven to prevent silencing of constitutive-expressing retroviral vectors in hiPSC. Tet-all-in-one retroviral vectors rely on a persistent expression of the transactivator and good regulation of the tet-responsive promoter. The incorporation of an additional element (with promoter activity) can interfere with the regulation. We incorporated elements of different sizes in sense and antisense transcriptional orientation into Tet-all-in-one gammaretroviral vectors and tested these for the regulation and anti-silencing capacity in different cell lines including murine and human iPSC. The U670 was identified to be the most potent element in preventing silencing which also conferred the strongest expression from the vector in the induced state. Longer fragments also sustained the gene expression but vector titers and induction efficiencies were impaired. Gammaretroviral Tet-all-in-one vectors turned out to be superior to their lentiviral counterparts due to the capability to produce higher titers. Finally, the expression and tight regulation of the thrombopoietin receptor Mpl within Tetinducible gammaretroviral vectors after incorporation of the U670s element was confirmed by cytokine-dependent cell growth. In addition, the PGK promoter could be replaced by the 670bp elements and sustained transgene silencing without interfering with the tight regulation of the vectors. With this part of the study, we found potent gammaretroviral vector candidates which pave the way for future application of overexpression of GATA1 and NFE2 in the human system to increase MK and platelet production in vitro.