Prostate cancer cells commonly spread through the circulation, but few successfully generate metastatic foci in bone. Osteoclastic cellular activity has been proposed as an initiating event for skeletal metastasis. Megakaryocytes (MKs) inhibit osteoclastogenesis, which could have an impact on tumor establishment in bone. Given the location of mature MKs at vascular sinusoids, they may be the first cells to physically encounter cancer cells as they enter the bone marrow. Identification of the interaction between MKs and prostate cancer cells was the focus of this study. K562 (human MK precursors) and primary MKs derived from mouse bone marrow hematopoietic precursor cells potently suppressed prostate carcinoma PC-3 cells in coculture. The inhibitory effects were specific to prostate carcinoma cells and were enhanced by direct cell-cell contact. Flow cytometry for propidium iodide (PI) and annexin V supported a proapoptotic role for K562 cells in limiting PC-3 cells. Gene expression analysis revealed reduced mRNA levels for cyclin D1, whereas mRNA levels of apoptosis-associated specklike protein containing a CARD (ASC) and death-associated protein kinase 1 (DAPK1) were increased in PC-3 cells after coculture with K562 cells. Recombinant thrombopoietin (TPO) was used to expand MKs in the marrow and resulted in decreased skeletal lesion development after intracardiac tumor inoculation. These novel findings suggest a potent inhibitory role of MKs in prostate carcinoma cell growth in vitro and in vivo. This new finding, of an interaction of metastatic tumors and hematopoietic cells during tumor colonization in bone, ultimately will lead to improved therapeutic interventions for prostate cancer patients. © 2011 American Society for Bone and Mineral Research.