B lymphocyte stimulator (BLyS) is a cytokine involved in differentiation and survival of follicular B cells along with humoral response potentiation. Lymphopenia is known to precipitate dramatic elevation in serum BLyS; however, the use of this effect to enhance humoral responses following vaccination has not been evaluated. We evaluated BLyS serum levels and antigen-specific antibody titers in 8 patients undergoing therapeutic temozolomide (TMZ)-induced lymphopenia, with concomitant vaccine against a tumor-specific mutation in the epidermal growth factor receptor (EGFRvIII). Our studies demonstrate that TMZ-induced lymphopenia corresponded with spikes in serum BLyS that directly preceded the induction of anti-EGFRvIII antigen-specific antibody titers, in some cases as high as 1:2,000,000. Our data are the first clinical observation of BLyS serum elevation and greatly enhanced humoral immune responses as a consequence of chemotherapy-induced lymphopenia. These observations should be considered for the development of future vaccination strategies in the setting of malignancy.