This review by Peng Du and Jun Wu provides a comprehensive overview of the hallmarks of totipotent and pluripotent stem cells, both in vivo and *in vitro*. The authors highlight the challenges and advancements in generating and characterizing these stem cells, particularly in the context of ethical and practical limitations. They discuss the dynamic states of pluripotent stem cells, including naïve, intermediate/formative, and primed states, and their molecular and functional characteristics. The review also explores the hallmarks of these states, such as signaling dependencies, gene expression patterns, DNA methylation, X chromosome inactivation, and metabolic features. Additionally, it covers assays for assessing the developmental potential of cultured stem cells, including blastocyst chimera formation, tetraploid complementation, and primordial germ cell specification. The authors emphasize the importance of understanding the differences between *in vivo* and *in vitro* conditions and the need for ongoing research to refine the characterization and application of these stem cells in various fields, including animal reproduction and regenerative medicine.This review by Peng Du and Jun Wu provides a comprehensive overview of the hallmarks of totipotent and pluripotent stem cells, both in vivo and *in vitro*. The authors highlight the challenges and advancements in generating and characterizing these stem cells, particularly in the context of ethical and practical limitations. They discuss the dynamic states of pluripotent stem cells, including naïve, intermediate/formative, and primed states, and their molecular and functional characteristics. The review also explores the hallmarks of these states, such as signaling dependencies, gene expression patterns, DNA methylation, X chromosome inactivation, and metabolic features. Additionally, it covers assays for assessing the developmental potential of cultured stem cells, including blastocyst chimera formation, tetraploid complementation, and primordial germ cell specification. The authors emphasize the importance of understanding the differences between *in vivo* and *in vitro* conditions and the need for ongoing research to refine the characterization and application of these stem cells in various fields, including animal reproduction and regenerative medicine.