Microglia are parenchymal macrophages that find their origin in the early embryonic yolk sac. These myeloid cells migrate to and take residency in the central nervous system (CNS), in which they are transcriptionally heterogenous in a context-dependent manner. Many microglial states and subtypes have been described by single-cell RNA sequencing (scRNA-seq), vastly accelerating knowledge of microglial biology; yet it is currently unknown how adult microglial heterogeneity emerges, or what regulons drive differentiation. Here, an atlas of transcriptional heterogeneity across the murine lifespan was developed, identifying 7 major myeloid subtypes in the CNS, one of which features an equivalent in human gestation. The occurrence of ex vivo-activated microglia (exAM) was also confirmed, a microglial state affected by cell isolation. In the present study it has been observed that standard scRNA-seq procedures introduce these effects, and that transcriptional inhibition is an effective means to their mitigation. This adaptation was implemented in a novel scRNA-seq protocol, generating a dataset in sexually immature microglia (SIM), a key developmental age for the acquisition of a mature microglial identity. Age was the main driver of microglial heterogeneity, albeit some evidence of sex-specific effects was also noted. Consequentially, male and female microglia display differential population dynamics, whereby age-associated subtypes emerge earlier in female mice. Furthermore, subtypespecific regulons in the female cortex were identified, offering novels ways to target and modify age-associated disease. To my knowledge, this study is the first that details microglial heterogeneity in health in a large-scale atlas, and the population dynamics that give rise to age-associated microglial heterogeneity.