AbstractMegakaryocytes (MKs) are the precursors of platelets (PLTs) and may be used for PLT production in vivo or in vitro, as well as a source for PLT-derived growth factors. Induced pluripotent stem cells represent an unlimited cell source for the in vitro production of MKs. This study aimed at developing an effective, xeno-free and scalable system to produce high numbers of MKs. In particular, microcarrier beads-assisted stirred bioreactors were evaluated as a means of improving MK yields. This method resulted in the production of 18.7 × 10⁷ MKs per 50 ml medium. Laminin-coated microcarriers increased MK production per iPSC by up to 10-fold. MKs obtained in this system showed typical features of mature MKs and were able to produce PLTs in vitro and in vivo. To increase safety, MKs produced in the bioreactors were irradiated; a procedure that did not affect their capability to form proPLTs and PTLs after transfusion. In vitro generated MKs represent a promising alternative to donor PLTs and open the possibility for the development of innovative MK-based cell therapies.