Pluripotent stem cells are able to form any terminally differentiated cell. They have opened new doors for experimental and therapeutic studies to understand early development and to cure degenerative diseases in a way not previously possible. Nevertheless, it remains important to resolve and define the mechanisms underlying pluripotent stem cells, as that understanding will impact strongly on future medical applications. The capture of pluripotent stem cells in a dish is bound to several landmark discoveries, from the initial culture and phenotyping of pluripotent embryonal carcinoma cells to the recent induction of pluripotency in somatic cells. On this developmental time line, key transcription factors, such as Oct4, Sox2 or Nanog, have been revealed not only to regulate but also to functionally induce pluripotency. These early master regulators of development control developmental signalling pathways that affect the cell cycle, regulate gene expression, modulate the epigenetic state and repair DNA damage. Besides transcription factors, microRNAs have recently been shown to play important roles in gene expression and are embedded into the regulatory network orchestrating cellular development. However, there are species-specific differences in pluripotent cells, such as surface marker expression and growth factor requirements. Such differences and their underlying developmental pathways require clear definition and have major impacts on the preclinical test bed of pluripotent cells.