The long-term maintenance of hematopoietic stem cells (HSCs) is assessed by serial bone marrow transplantation (BMT), in which HSCs are injected intravenously. Recently, we have found that intra-bone marrow (IBM)–BMT can efficiently reconstitute the hematopoietic system with cells of donor origin, in contrast to conventional intravenous (IV) BMT. In the present study, we have compared the long-term maintenance of HSCs using multiple rounds of serial IV-BMT and IBM-BMT. The frequencies of donor-derived progenitor cells (Lin⁻ /c-kit⁺ cells) and more primitive progenitors (Lin⁻ /c-kit⁺/CD34⁺ /Sca-1⁺ cells) were higher in the tertiary recipients by serial IBM-BMT than in those that had received bone marrow cells by serial IV-BMT. Furthermore, neither donor-derived progenitor cells nor mature hematolymphoid cells were detected in ∼25% of the tertiary recipients after serial IV-BMT, indicating that progenitor cells can be efficiently maintained by IBM-BMT but not by IV-BMT. Finally, we confirmed that the recipients treated with the primary IBM-BMT (without carrying out serial BMT) showed a significantly higher survival rate than those treated with IV-BMT. These findings clearly show that IBM-BMT efficiently promotes the longterm maintenance of donor-derived hematopoiesis.