Mediation analysis is a statistical approach that can provide insights regarding the intermediary processes by which an intervention or exposure affects a given outcome. Mediation analyses rose to prominence, particularly in social science research, with the publication of Baron and Kenny’s seminal paper and is now commonly applied in many research disciplines, including health services research. Despite the growth in popularity, applied researchers may still encounter challenges in terms of conducting mediation analyses in practice. In this paper, we provide an overview of conceptual and methodological challenges that researchers face when conducting mediation analyses. Specifically, we discuss the following key challenges: (1) Conceptually differentiating mediators from other ’third variables,’ (2) Extending beyond the single mediator context, (3) Identifying appropriate datasets in which measurement and temporal ordering support the hypothesized mediation model, (4) Selecting mediation effects that reflect the scientific question of interest, (5) Assessing the validity of underlying assumptions of no omitted confounders, (6) Addressing measurement error regarding the mediator, and (7) Clearly reporting results from mediation analyses. We discuss each challenge and highlight ways in which the applied researcher can approach these challenges.