Phosphoinositides (PI) are a group of cellular phospholipids of the utmost importance for the regulation of cellular physiology and membrane dynamics. In eukaryotic cells they are generated by phosphorylation of phosphatidylinositol (PtdIns) by activation of a series of kinases and phosphatases. In this study we have investigated the expression and localization of FYVE and PX domains that specifically bind phosphatidylinositol 3-monophosphate [PI(3)P] and their impact on the development of megakaryocytes and production of proplatelets. We have cloned fluorescently labeled tandem FYVE and mutated domain (eGFP-2x-FYVE and eGFP-2x-FYVE mut.) into a retroviral vector. YFP-PX domain already cloned in retroviral vector was also mutated. By immunofluorescence, a specific localization pattern of eGFP-2x-FYVE was observed in form of discrete dots throughout the cell. These dots colocalized with EEA1 (early endosome marker), LAMP1 (late endosome marker) and LysoTracker (lysosome marker). YFP-PX was also characterized by the appearance of discrete vesicles partially colocalizing with EEA1. Both mutated domains lost the distinctive pattern of localization and appeared as a uniform signal throughout the cell. Selective inhibition of PI3-kinase class III which produces PI(3)P led to a disturbed pattern of eGFP-2x-FYVE localization, and thus confirmed specificity of the cloned domain for PI(3)P. Megakaryocytes that express GFP-2x-FYVE produce significantly less proplatelets than when expressing only eGFP or mutated domain. Finally, a colocalization of PI(3)P binding domains (eGFP-2x-FYVE, YFP-PX) with protein kinase A was determined, implicating a potentially new role of PI(3)P. These results indicate a high significance of FYVE and PX domains, create new questions, and elicit new research directions.