Male sterility is a relatively common consequence of cancer treatment. For prepubertal boys with cancer, fertility preservation can theoretically be achieved by freezing a small testis biopsy prior to cancer treatment, followed by in vitro propagation of spermatogonial stem cells (SSCs) from this biopsy and autotransplanting these SSCs after cure of cancer. Avoiding the reintroduction of any malignant cells present in this biopsy is of critical importance before clinical application of SSCs autotransplantation can be further explored. Here, we show that ALL cells cultured separately in medium used to propagate human SSCs did not survive beyond 14 days of culture. We also demonstrate that irrespective of their initial concentrations (0. 04%, 0. 4%, 4% and 40%), ALL cells mixed with testicular cells were undetectable beyond 18-26 days of culture as measured by highly-sensitive (1: 10, 000 to 1: 100, 000 cells) antigen-receptor PCR (MRD-PCR). These results prove that our culture system not only allows for efficient propagation of SSCs but also eliminates contaminating malignant ALL cells.