Homeodomain transcription factors play important roles in the specification and differentiation of neuronal subpopulations. In the cerebral cortex, the expression patterns of Cux-1 and Cux-2 in the medial ganglionic eminence (MGE) suggest a role for these transcription factors in the development of interneurons, a heterogeneous neuronal population. In this report, we describe expression of Cux-1 and Cux-2 proteins in Reelin-secreting interneurons of the cortical plate, but not in calretinin or parvalbumin subpopulations. The role of Cux genes in the development of Reelin positive neurons was studied using Cux-1 and Cux-2 knockout mice. These experiments demonstrate that Cux-1-/-; Cux-2-/- double mutation is embryonically lethal. Although this phenotype is highly penetrant, a small proportion of mice develop to birth (P0). Analysis of these animals demonstrate that expression of Reelin is completely absent in layers II-IV of Cux-1-/-; Cux-2-/- double mutant mice, but it is not affected in the cortex of Cux-1-/- or Cux-2-/- single mutants. No Cux-1-/-; Cux-2-/- double-mutant were collected after P0. Since, GABA-ergic populations mature at late postnatal stages, this did not allow us to analyze the expression of subclass specific markers and define the affected interneuron subpopulations. Our analysis of Cux-1-/-; Cux-2-/- double mutant thus demonstrates essential yet redundant roles for Cux-1 and Cux-2 in specifying Reelin expressing cortical interneurons.