A Culex quinquefasciatus colony was established from surviving late instars and pupae from a semifield microcosm test with Natular® XRG (a granular formulation containing 2.5% spinosad) applied at 14.2 kg/ha. The initial lethal concentrations for 50% and 90% of the population (LC₅₀ and LC₉₀) against Natular XRG were determined in the laboratory for the 1st generation progeny (F₁). Selection was applied at LC_70–90 levels to 10,000–15,000 of late 3rd and early 4th instars each generation. Susceptibility changes were determined every other generation and referenced to a susceptible laboratory colony as well as freshly collected wild populations of the same species concurrently. Tolerance to spinosad (resistance ratio <5.0 fold) was observed up to F₈ generation in the selected population. Thereafter, resistance ratios increased significantly from F₁₀ to F₁₆ generations, being 7.26- to 20.52-fold at the LC₅₀ and 7.48- to 20.08-fold at the LC₉₀ level.