Understanding the molecular pathogenesis of Coxiella burnetii, the causative agent of human Q fever, has historically been hindered by the technical difficulties of genetically manipulating obligate intracellular bacteria. The recent development of culture conditions suitable for axenic propagation of C. burnetii has paved the way for the application of a range of genetic techniques to address key questions within the field. Recent studies using mutational analysis have revealed that the C. burnetii Dot/Icm type 4 secretion system (T4SS) is an important virulence determinant that is essential for renovation of a lysosome into a mature Coxiella-containing vacuole (CCV) permissive of intracellular replication. Interestingly, a mutant of C. burnetii deficient in Dot/Icm function was found to be capable of replicating within the parasitophorous vacuole created by Leishmania amazonensis, which indicates that C. burnetii replication is not dependent on the cohort of Dot/Icm effector proteins per se but rather that the collective actions of effectors are required to create the specialized niche supportive of replication. Thus, a role for the Dot/Icm T4SS during the intracellular life cycle of C. burnetii has been more clearly defined by these studies, which demonstrate that advances in genetic analysis should allow future studies to focus on the intricacies of Dot/Icm effector functions that facilitate development of the unique CCV.