How Kit expression is regulated in the germline is still an open question. SOHLH1 and SOHLH2, two bHLH transcription factors specifically expressed in germ cells, have been recently involved in the differentiation of spermatogonia and oocytes. In the male, deletion of each transcription factor leads to the disappearance of Kit-expressing spermatogonia in the prepuberal testis. In the female, both SOHLH1 and SOHLH2 ablations are responsible of oocyte loss in the neonatal ovary. To investigate if Kit expression might be regulated by these two transcription factors in male germ cells, we have first performed an expression study of SOHLH1 and SOHLH2 during fetal and postnatal development. In these experiments we found a strong positive correlation between Kit and the two transcription factors only in postnatal spermatogonia. SOHLH2 was found enriched in undifferentiated spermatogonia, whereas SOHLH1 expression was maximal in Kit-dependent stages. Expression of SOHLH1, but not SOHLH2, was increased in postnatal mitotic germ cells by treatment with All-trans Retinoic Acid (AtRA), an analog of retinoic acid. We also found that E-box sequences contained within the Kit promoter and its first intron can be transactivated in transfection experiments overexpressing either Sohlh1or Sohlh2. Co-transfection of both factors showed a cooperative effect. EMSA experiments further showed that SOHLH1 and SOHLH2 can independently and cooperatively bind an E-box containing probe. In vivo, co-immunoprecipitation results evidenced that the two proteins interact and overexpression of both factors increases endogenous Kit expression in ES cells. Finally, by ChIP analysis, SOHLH1 was found to occupy an E-box containing region within kit promoter in spermatogonia chromatin. Altogether, our results suggest that SOHLH1 and SOHLH2 directly stimulate Kit transcription in postnatal spermatogonia, thus activating the signalling involved in spermatogonia differentiation and spermatogenetic progression.