The HIV-1 Gag protein assembles into immature capsids when expressed in human cells. Although self-assembly of Gag was once thought to be sufficient to explain capsid formation, in the past decade it has become increasingly apparent that in cells, the pathway from Gag synthesis to assembled capsids is coordinated and facilitated by host factors. These cellular factors likely direct the trafficking, membrane targeting, and multimerization of Gag, and could also assist with encapsidation of viral RNA. While some of these factors have been identified, much remains to be learned about the mechanisms by which they act to promote capsid formation. Moreover, studies suggest that the amount of intracellular Gag undergoing assembly per se at any given time may be quite low, with the majority of Gag in some cell types undergoing degradation or representing Gag that remains cell-associated after assembly. If this model holds true, then defining the Gag subpopulations on which individual cellular factors act will be important for understanding the role of host factors. Towards this end, it will be important to find markers and features that can distinguish subpopulations of Gag destined for different outcomes so that these populations can be quantified and tracked separately both at the biochemical and microscopic level. Thus, the challenge for the future will be to understand which cellular factors act during the pathway from Gag synthesis to assembly, and exactly where and how they act in this pathway. (AIDS Rev. 2007;9:150-61) Corresponding author: Jaisri R. Lingappa, jais@u.washington.edu