Fibronectin (FN) plays important roles in the proliferation, differentiation, and maintenance of megakaryocytic-lineage cells through FN receptors. However, substantial role of FN receptors and their functional assignment in proplatelet-like formation (PPF) of megakaryocytes are not yet fully understood. Herein, we investigated the effects of FN receptors on PPF using the CHRF-288 human megakaryoblastic cell line, which expresses VLA-4 and VLA-5 as FN receptors. FN and phorbol 12-myristate 13-acetate (PMA) were essential for inducing PPF in CHRF-288 cells. Blocking experiments using anti-β1-integrin monoclonal antibodies indicated that the adhesive interaction with FN via VLA-4 and VLA-5 were required for PPF. PPF induced by FN plus PMA was accelerated when CHRF-288 cells were enforced adhering to FN by TNIIIA2, a peptide derived from tenascin-C, which we recently found to induce β1-integrin activation. Adhesion to FN enhanced PMA-stimulated activation of extracellular signal-regulated protein kinase 1 (ERK1)/2 and enforced adhesion to FN via VLA-4 and VLA-5 by TNIIIA2-accelerated activation of ERK1/2 with FN plus PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38, and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN plus PMA, even with TNIIIA2. Thus, the enhanced activation of ERK1/2 by FN, PMA plus TNIIIA2 was responsible for acceleration of PPF with FN plus PMA.