For decades, controversy surrounded the question of whether spontaneous lateral mixing of membranes into coexisting liquid phases could organize proteins and liquids on micron scales within unperturbed, living cells. A clear answer hinged on the observation of hallmarks of a reversible phase transition. In this paper, by directly imaging micron-scale membrane domains of yeast vacuoles both in vivo and cell-free, we demonstrated that the domains arise through a phase-separation mechanism. Domains in yeast vacuole membranes are large, have smooth boundaries, and can merge quickly, consistent with fluid phases. Moreover, the domains disappear above a distinct miscibility transition temperature and reappear below it, over multiple heating a cooling cycles. Hence, large-scale membrane organization in living cells under physiologically-relevant conditions can be controlled by tuning a single thermodynamic parameter. This paper exemplifies the collaborative research culture of the University of Washington. Sarah and Alex (a professor in the Department of Biochemistry) were joint corresponding authors; Scott and Glennis were joint first authors. To read more about this work, download out the article that Physics Today wrote about our discovery, or check out Alex's tweetorial.