TLF (TBP-like factor) is a protein commonly thought to belong to the general transcription initiation complex. TLF is evolutionarily conserved and has been shown to be essential for early development in C. elegans, zebrafish and Xenopus. In mammals however, TLF has a specialised function, as revealed by targeted mutation of the gene in the mouse germline. The TLF mutation elicits a complete arrest of late spermiogenesis and increased haploid cell apoptosis. We explored in more detail the molecular function that TLF plays in the differentiation program of male germ cells. A comparison of TBP and TLF reveals drastic differences, both in their temporal expression pattern and in their intracellular location. While TBP is ubiquitously expressed, TLF expression is strictly developmentally regulated, being very high in late pachytene spermatocytes, suggesting a function prior to the apoptosis of the haploid cells. A refined study of TLF-deficient mice reveals defective acrosome formation in early stage spermatids. Most importantly, our results uncover an unsuspected function of TLF in chromatin organisation. Indeed, early spermatids in TLF-deficient mice display a fragmentation of the chromocenter, a condensed structure formed by the association of centromeric heterochromatin and containing the HP1 proteins. This defect is likely to be the primary cause of spermatogenic failure in the TLF mutant mice.