The documentation of gendered disparities in health has led to calls for scientists across the spectrum of biomedical research to attend to sex and gender considerations in their work. In basic experimental laboratory research with cells, it has often been suggested that researchers should use cells from both male and female donors in their experiments. In this essay, I discuss some of the complexities of addressing sex in vitro that render this a more complicated proposition than it may first appear. In fact, given that “sex” involves manifestations across multiple levels of organization, I argue that it may not actually be sensible to think of isolated cells as “having” a sex at all. Cells themselves aside, the conditions under which cells are grown in the laboratory are not able to comprehensively model the complexities of sex. Finally, I question the utility of male-female comparisons for addressing sex, exploring some of the implications of treating sex as though it were a simple independent variable. Ultimately, I contend that addressing sex in cell culture systems demands sophisticated theorizing, lest we inadvertently play into essentialist and biological determinist discourses of sex and gender. It is not enough to simplistically include both female and male cells in in vitro research. We must also attend to the material realities of laboratory practice and enrich scientific discourses of sex and gender if we are to develop appropriate, nuanced approaches to addressing issues related to sex/gender in cell culture research.