PATZ1 is a transcriptional factor downregulated in thyroid cancer whose re-expression in thyroid cancer cells leads to a partial reversion of the malignant phenotype, including the capacity to proliferate, migrate, and undergo epithelial-to-mesenchymal transition. We have recently shown that PATZ1 is specifically downregulated downstream of the Ras oncogenic signaling through miR-29b, and that restoration of PATZ1 in Ha-Ras transformed FRTL5 rat thyroid cells is able to inhibit their capacities to proliferate and migrate in vitro. Here, we analyzed the impact of PATZ1 expression on the in vivo tumorigenesis of these cells. Surprisingly, FRTL5-Ras-PATZ1 cells showed enhanced tumor initiation when engrafted in nude mice, even if their tumor growth rate was reduced compared to that of FRTL5-Ras control cells. To further investigate the cause of the enhanced tumor engraftment of FRTL5-Ras-PATZ1 cells, we analyzed the stem-like potential of these cells through their capacity to grow as thyrospheres. The results showed that restoration of PATZ1 expression in these cells increases stem cell markers’ expression and self-renewal ability of the thyrospheres while limiting their growth capacity. Therefore, we suggest that PATZ1 may play a role in enhancing the stem cell potential of thyroid cancer cells, but, at the same time, it impairs the proliferation of non-stem cells.