The multifunctional proteins leukemia inhibitory factor (LIF) and insulin-like growth factor I (IGF-I) are expressed in overlapping patterns during development and, therefore, may act cooperatively. We show that mice doubly deficient in LIF and IGF-I all died at birth of apparent respiratory failure. Growth retardation, muscle hypoplasia and delayed ossification in IGF-I-deficient E18.5 mice were exacerbated by the absence of LIF. The transcription factor Sp3 was decreased in the skeleton of the double null mice. Pronounced depletion of olfactory bulb neurons, in contrast, was only IGF-I-dependent. The lungs displayed reduced air space in the IGF-I-deficient embryos and neonates, phenotype exacerbated in the double nulls, which showed abnormal epithelial cells and decreased Sp3 expression. In addition, the transcription factor TTF-1 and the surfactant protein B were lower in the lung of the double null neonates than in all other genotypes. LIF and IGF-I, thus, have cooperative and distinct tissue functions during development. Their essential role in bone ossification apparently involves Sp3, and in lung maturation Sp3 together with TTF-1.