We have previously reported the effects of 2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a synthetic phospholipid, on megakaryocytic differentiation of myeloid leukemia cells. Here, we demonstrate that (R)-TEMOSPho enhances megakaryopoiesis and plateletogenesis from primary hematopoietic stem cells (HSCs) induced by thrombopoietin (TPO). Specifically, we demonstrate at sub-saturation levels of TPO, the addition of (R)-TEMOSPho enhances differentiation and maturation of megakaryocytes (MKs) from murine HSCs derived from fetal liver. Furthermore, we show that production of platelets with (R)-TEMOSPho in combination with TPO is also more efficient than TPO alone and that platelets generated in vitro with these two agents are as functional as those from TPO alone. TPO can thus be partly replaced by or supplemented with (R)-TEMOSPho, and this in turn implies that (R)-TEMOSPho can be useful in efficient platelet production in vitro and potentially be a valuable option in designing cell-based therapy.