Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.