It has been a century since the first report showed that vaccination of animals with embryonic tissues could prevent the outgrowth of tumours. Subsequent studies have confirmed that immune cells, in particular T cells, can be primed against multiple types of tumour by embryonic tissues and pluripotent stem cells (PSCs). The rationale for using PSCs is based on the overlap in transcriptome profiles between these cells and tumours. Moreover, current evidence suggests PSCs may display similar antigens to those presented by cancer stem cells/cancer-initiating cells. Hence, PSC-based vaccines are proposed to aid in both the prevention of tumour initiation and tumour recurrence. However, the immunogenicity of PSCs remains controversial and the precise antigens that contribute to their immunogenicity are unknown. Therefore, we have initiated a mass spectrometry-based epitope discovery program, interrogating the HLA-I immunopeptidome of human PSCs to define the mechanisms that enable PSCs to stimulate anti-cancer immune responses. Current work moves to explore the naturally presented HLA‐I immunopeptidome of PSCs to determine similarities and differences with cancerous and healthy human tissues. These studies will help define the features of PSCs that stimulate anti-cancer responses, informing the design of cancer vaccines.