Spatial and temporal cues govern the genesis of a diverse array of neurons located in the dorsal spinal cord, including dI1-dI6, dILA, and dILBsubtypes, but their physiological functions are poorly understood. Here we generated a new line of conditional knock-out (CKO) mice, in which the homeobox geneTlx3was removed in dI5 and dILBcells. In theseCKOmice, development of a subset of excitatory neurons located in laminae I and II was impaired, including itch-related GRPR-expressing neurons, PKCγ-expressing neurons, and neurons expressing three neuropeptide genes: somatostatin, preprotachykinin 1, and the gastrin-releasing peptide. TheseCKOmice displayed marked deficits in generating nocifensive motor behaviors evoked by a range of pain-related or itch-related stimuli. The mutants also failed to exhibit escape response evoked by dynamic mechanical stimuli but retained the ability to sense innocuous cooling and/or warm. Thus, our studies provide new insight into the ontogeny of spinal neurons processing distinct sensory modalities.