With recent approval of the first dendritic cell (DC) vaccine for patient use, many other DC vaccine approaches are now being tested in clinical trials. Many of these DC vaccines employ tumor cell lysates (TL) generated from cells cultured in atmospheric oxygen (~20% O 2) that greatly exceeds levels found in tumors in situ. In this study, we tested the hypothesis that TLs generated from tumor cells cultured under physiologic oxygen (~5% O 2) would be more effective as a source for DC antigens. Gene expression patterns in primary glioma cultures established at 5% O 2 more closely paralleled patient tumors in situ and known immunogenic antigens were more highly expressed. DCs treated with TLs generated from primary tumor cells maintained in 5% O 2 took up and presented antigens to CD8 T cells more efficiently. Moreover, CD8 T cells primed in this manner exhibited superior tumoricidal activity against target cells cultured in either atmospheric 20% O 2 or physiologic 5% O 2. Together, these results establish a simple method to greatly improve the effectiveness of DC vaccines in stimulating the production of tumoricidal T cells, with broad implications for many of the DC-based cancer vaccines being developed for clinical application.