Stem cells are the cells which can give rise to multiple different cell types. There are two main types of stem cells present in the mammalian system: Embryonic stem cells (ESCs) and Adult stem cells. These two cell types give rise to myriads of cell types during various stages of embryonic and adult development. Embryonic Stem cells, which are derived from the inner cell mass of blastocyst stage embryo, possess two unique characteristics of self-renewal and pluripotency. Self-renewal is the ability to go through numerous cycles of cell division while maintaining the undifferentiated state and pluripotency is the ability to give rise to any of the differentiated cell types obtained from three germ layers. Stem cells share this unlimited proliferation capacity with the cancer cells, which are considered as rogue cells of any mammalian system. While the unlimited self-renewal capacity of stem cells works beneficially towards a biological system and is constructive or regenerative in nature, the unlimited proliferation capacity of cancer cells is harmful. Because of this unlimited proliferation capacity, cancer cells proliferate independent of external growth signals, show resistance to apoptosis, evasion of anti-growth signals and immune destruction. Although stem cells are also resistant to apoptosis, however, they are not involved in immune destruction. Over the last few decades, enough data has accumulated how extrinsic and intrinsic factors are involved in regulating the unique characteristics of stem cells and unlimited proliferation capacity of cancer cells and cancer stem cells. Since, reactive oxygen species are involved in regulating many physiological and biochemical functions in a cell. This chapter discusses how reactive oxygen species are involved in regulating self-renewal and pluripotency of stem cells and impacts the unique features of cancer cells and cancer stem cells.