The LIM homeodomain transcription factor Lmx1b has been suggested to be required for the differentiation of midbrain dopaminergic (mDA) neurons. However, whether the loss of mDA neurons in Lmx1b(-/-) mice is due to its intrinsic role in the mDA lineage or to a consequence of the malformations caused by the earlier mid/hindbrain patterning defects remains to be clarified. We report here that Lmx1b expression in mDA neurons is dispensable for their differentiation and maintenance, and the loss of mDA neurons in Lmx1b(-/-) mice is due to the disruption of inductive activity of the isthmic organizer (IsO) in the absence of Lmx1b at the mid/hindbrain boundary (MHB). We found that mDA neurons revealed by tyrosine hydroxylase (TH), Pitx3, Nurr1, and dopamine transporter were indistinguishable from wild-type controls during embryonic development as well as in adulthood in TH-Cre;Lmx1b(flox/-) and Dat(Cre/+);Lmx1b(flox/-) mice, in which Lmx1b was selectively deleted in differentiating mDA neurons. In addition, mDA neurons were recovered in Lmx1b(-/-) mice, when IsO activity was restored by Wnt1-Lmx1b transgene at MHB. The restored IsO activity was evidenced by apparently normal tectum and cerebellum and recurrence of expression of Fgf8 and Wnt1 at MHB in Wnt1(Lmx1b);Lmx1b(-/-). Furthermore, when Lmx1b was deleted in the whole brain after the formation of IsO by Nestin-Cre, mDA neurons were normal, whereas serotonergic neurons displayed defective development phenocopying what observed in Lmx1b(-/-) mice. Thus, our results indicate that the inductive activity of IsO is essential, but Lmx1b expression in mDA neurons is dispensable for their differentiation and maintenance.