In vitro cultivation of Plasmodium falciparum, the agent of severe human malaria, has enabled advances in basic research and accelerated the development of new therapies. Since the introduction of in vitro parasite culture nearly 40 years ago, most workers have used a medium consisting of RPMI 1640 medium supplemented with lipids and hypoxanthine. While these standardized conditions yield robust parasite growth and facilitate comparison of results from different studies, they may also lead to implicit assumptions that limit future advances. Here, I review recent studies that used modified culture conditions to challenge these assumptions and explore parasite physiology. The findings are relevant to understanding in vivo parasite phenotypes and the prioritization of antimalarial targets.