In vivo administration of soluble Flt3L increases dendritic cell (DC) numbers to favor improved DC targeting of vaccine antigens, augmenting vaccine efficiency. In addition to confirming the effectiveness of human Flt3L in macaques, we strove to determine the optimal regimen to elevate numbers of functional DCs. Circulating DCs were identified within lineage-human leukocyte antigen-DR+ cells, which comprised CD11c-CD123+ plasmacytoid DCs (PDCs) and CD123- cells including CD11c+ CD123- myeloid DCs as well as CD11c- CD123- cells. Traditionally, DCs have been monitored 1-2 days after 10- to 14-day treatments with Flt3L (100 µg/kg/day). We demonstrate that although standard treatment increased macaque DC percentages, as little as 5-7 days of treatment was sufficient, if not more effective at mobilizing DCs. Moreover, DC frequency continued to escalate over the ensuing days, peaking at ~4 days post 7 days of treatment and ultimately decreasing thereafter. As expected, there was a more pronounced increase in the percentages and actual numbers of CD123- cells (CD11c+ and CD11c- subsets) compared with PDCs. Flt3L-mobilized DCs exhibited slightly increased CD80/CD86 expression but typically still that of immature DCs and were resilient to freeze-thawing. Overnight culture activated the cells, up-regulating CD80/CD86 expression as well as interleukin-12 release, typically being boosted by CD40L. This was even more apparent for enriched DC cultures. These data verify that peak mobilization of large numbers of functional macaque DCs occurs a few days, not immediately, after short-term Flt3L dosing. This has important implications for improved DC-targeting vaccine strategies to prevent infection with human immunodeficiency virus and other pathogens.