Although the differentiation of pluripotent cells in embryonic stem cells (ESCs) is often associated with protein kinase-mediated signaling pathways and Tousled-like kinase 1 (Tlk1) is required for development in several species, the role of Tlk1 in ESC function remains unclear. Here, we used mouse ESCs to study the function of Tlk1 in pluripotent cells. The knockdown (KD)-based Tlk1-deficient cells showed that Tlk1 is not essential for ESC self-renewal in an undifferentiated state. However, Tlk1-KD cells formed irregularly shaped embryoid bodies and induced resistance to differentiation cues, indicating their failure to differentiate into an embryoid body. Consistent with their failure to differentiate, Tlk1-KD cells failed to downregulate the expression of undifferentiated cell markers including Oct4, Nanog, and Sox2 during differentiation, suggesting a negative role of Tlk1. Interestingly, Tlk1 overexpression sufficiently downregulated the expression of core pluripotency factors possibly irrespective of its kinase activity, thereby leading to a partial loss of self-renewal ability even in an undifferentiated state. Moreover, Tlk1 overexpression caused severe growth defects and G2/M phase arrest as well as apoptosis. Collectively, our data suggest that Tlk1 negatively regulates the expression of pluripotency factors, thereby contributing to the scheduled differentiation of mouse ESCs.